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Publication numberUS7511190 B2
Publication typeGrant
Application numberUS 10/374,780
Publication dateMar 31, 2009
Filing dateFeb 25, 2003
Priority dateNov 17, 1999
Fee statusPaid
Also published asUS8110725, US20040019927, US20060162006, US20090276912, US20120144518
Publication number10374780, 374780, US 7511190 B2, US 7511190B2, US-B2-7511190, US7511190 B2, US7511190B2
InventorsRobert A. Creelman, Oliver Ratcliffe, T. Lynne Reuber, James Zhang, Gregory Nadzan
Original AssigneeMendel Biotechnology, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Expressing a polypeptide encoded by a preferred polynucleotide sequence in a plant and detecting at least one factor that is modulated by or interacts with such polypeptide
US 7511190 B2
Abstract
The invention relates to plant transcription factor polypeptides, polynucleotides that encode them, homologs from a variety of plant species, and methods of using the polynucleotides and polypeptides to produce transgenic plants having advantageous properties compared to a reference plant. Sequence information related to these polynucleotides and polypeptides can also be used in bioinformatic search methods and is also disclosed.
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Claims(35)
1. A transgenic plant that has more chlorophyll than a non-transformed control plant or a wild-type control plant of the same species, wherein the transgenic plant comprises a recombinant polynucleotide sequence encoding a transcription factor polypeptide that has at least 95% amino acid sequence identity to SEQ ID NO: 304.
2. The transgenic plant of claim 1, wherein the transcription factor polypeptide has at least 98% amino acid sequence identity to SEQ ID NO: 304.
3. The transgenic plant of claim 1, wherein the transcription factor polypeptide comprises SEQ ID NO: 304.
4. The transgenic plant of claim 1, wherein the recombinant polynucleotide sequence is operably linked to a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
5. The transgenic plant of claim 1, wherein the transcription factor polypeptide is expressed in the transgenic plant at levels sufficient to increase chlorophyll a in the transgenic plant as compared to the non-transformed plant or the wild-type control plant of the same species.
6. The transgenic plant of claim 1, wherein the transcription factor polypeptide is expressed in the transgenic plant at levels sufficient to increase chlorophyll b in the transgenic plant as compared to the non-transformed plant or the wild-type control plant of the same species.
7. The transgenic plant of claim 1, wherein the transgenic plant has increased photosynthetic capacity relative to the non-transformed plant or the wild-type control plant of the same species.
8. A transgenic plant that has greater photosynthetic capacity than a non-transformed control plant or a wild-type control plant of the same species, wherein the transgenic plant comprises a recombinant polynucleotide sequence encoding a transcription factor polypeptide that has at least 95% amino acid sequence identity to SEQ ID NO: 304.
9. The transgenic plant of claim 8, wherein the transcription factor polypeptide has at least 98% amino acid sequence identity to SEQ ID NO: 304.
10. The transgenic plant of claim 8, wherein the transcription factor polypeptide comprises SEQ ID NO: 304.
11. The transgenic plant of claim 8, wherein the recombinant polynucleotide sequence is operably linked to a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
12. A transformed plant seed, wherein a transgenic plant grown from the transformed plant seed has more chlorophyll and/or greater photosynthetic capacity than a non-transformed control plant or a wild-type control plant of the same species, and the transformed plant seed comprises:
a recombinant polynucleotide sequence encoding a transcription factor polypeptide that has at least 95% amino acid sequence identity to SEQ ID NO: 304.
13. The transformed plant seed of claim 12, wherein the transcription factor polypeptide has at least 98% amino acid sequence identity to SEQ ID NO: 304.
14. The transformed plant seed of claim 12, wherein the transcription factor polypeptide comprises SEQ ID NO: 304.
15. The transformed plant seed of claim 12, wherein the recombinant polynucleotide sequence is operably linked to a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
16. A transgenic plant that comprises a recombinant polynucleotide sequence encoding a transcription factor polypeptide comprising SEQ ID NO: 304, wherein the transgenic plant stably expresses the transcription factor polypeptide.
17. The transgenic plant of claim 16, wherein the recombinant polynucleotide sequence is operably linked to a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
18. The transgenic plant of claim 16, wherein the transcription factor polypeptide is expressed in the transgenic plant at levels sufficient to increase carotenoid levels in the transgenic plant as compared to a non-transformed plant or a wild-type control plant of the same species.
19. The transgenic plant of claim 16, wherein the transcription factor polypeptide is expressed in the transgenic plant at levels sufficient to increase chlorophyll a in the transgenic plant as compared to a non-transformed plant or a wild-type control plant of the same species.
20. The transgenic plant of claim 16, wherein the transcription factor polypeptide is expressed in the transgenic plant at levels sufficient to increase chlorophyll b in the transgenic plant as compared to a non-transformed plant or a wild-type control plant of the same species.
21. The transgenic plant of claim 16, wherein the transgenic plant has increased photosynthetic capacity relative to a non-transformed plant or a wild-type control plant of the same species.
22. The transgenic plant of claim 16, wherein the transgenic plant is a transformed seed that comprises the recombinant polynucleotide sequence of claim 16.
23. A method for producing a transgenic plant with more chlorophyll than a non-transformed plant or a wild-type control plant of the same species, the method steps consisting of:
a. transforming a target plant with an expression vector to produce the transgenic plant, wherein the expression vector comprises a polynucleotide sequence that encodes a transcription factor polypeptide that has at least 95% amino acid sequence identity to SEQ ID NO: 304; and
b. growing the transgenic plant, wherein the transcription factor polypeptide is expressed in the transgenic plant at levels sufficient to produce more chlorophyll in the transgenic plant as compared to the non-transformed or the wild-type control plant.
24. The method of claim 23, wherein the transcription factor polypeptide has at least 98% amino acid sequence identity to SEQ ID NO: 304.
25. The method of claim 23, wherein the transcription factor polypeptide comprises SEQ ID NO: 304.
26. The method of claim 23, wherein the methods steps further comprise:
c. selecting the transgenic plant having more chlorophyll than the non-transformed or the wild-type control plant.
27. The method of claim 23, wherein the expression vector comprises a constitutive promoter, an inducible promoter, or a tissue-specific promoter operably linked to the polynucleotide sequence.
28. A method for producing a transgenic plant with an increased photosynthetic capacity relative to a non-transformed plant or a wild-type control plant of the same species, the method steps consisting of:
a. transforming a target plant with an expression vector to produce the transgenic plant, wherein the expression vector comprises a polynucleotide sequence that encodes a transcription factor polypeptide that has at least 95% amino acid sequence identity to SEQ ID NO: 304; and
b. growing the transgenic plant, wherein the transcription factor polypeptide is expressed in the transgenic plant at levels sufficient to increase the photosynthetic capacity in the transgenic plant as compared to the photosynthetic capacity in the non-transformed or the wild-type control plant.
29. The method of claim 28, wherein the transcription factor polypeptide has at least 98% amino acid sequence identity to SEQ ID NO: 304.
30. The method of claim 28, wherein the transcription factor polypeptide comprises SEQ ID NO: 304.
31. The method of claim 28, wherein the methods steps further comprise:
c. selecting the transgenic plant having the increased photosynthetic capacity relative to the non-transformed or the wild-type control plant.
32. The method of claim 28, wherein the expression vector comprises a constitutive promoter, an inducible promoter, or a tissue-specific promoter operably linked to the polynucleotide sequence.
33. A transgenic plant that comprises a recombinant polynucleotide sequence encoding a transcription factor polypeptide that has at least 95% amino acid sequence identity to SEQ ID NO: 304;
wherein the transgenic plant stably expresses the transcription factor polypeptide.
34. The transgenic plant of claim 33, wherein the transcription factor polypeptide has at least 98% amino acid sequence identity to SEQ ID NO: 304.
35. The transgenic plant of claim 33, wherein expression of the recombinant polynucleotide sequence is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.
Description
RELATIONSHIP TO COPENDING APPLICATIONS

This application is a continuation-in-part of application Ser. No. 09/713,994, filed Nov. 16, 2000, which claims the benefit of Application No. 60/166,228, filed Nov. 17, 1999, which also claims the benefit of Application No. 60/197,899, filed Apr. 17, 2000, and also claims the benefit of Application No. 60/227,439, filed Aug. 22, 2000. This application is a continuation-in-part of Application No. 09/934,455, filed Aug. 22, 2001, abandoned, which claims the benefit of Application No. 60/227,439, filed Aug. 22, 2000. Application Ser. No. 09/934,455, abandoned, is also a continuation-in-part of application Ser. No. 09/713,994, filed Nov. 16, 2000 and also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001, abandoned. This application is a continuation-in-part of application Ser. No. 10/225,068, filed Aug. 9, 2002, which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001. Application Ser. No. 10/225,068 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001, abandoned, and also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002, abandoned. This application is a continuation-in-part of application Ser. No. 10/225,066, filed Aug. 9, 2002, which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11, 2001. Application Ser. No. 10/225,066 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001, abandoned, and also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002, abandoned. This application is a continuation-in-part of application Ser. No. 10/225,067, filed Aug. 9, 2002, which claims the benefit of Application No. 60/310,847, filed Aug. 9, 2001, which also claims the benefit of Application No. 60/336,049, filed Nov. 19, 2001, and also claims the benefit of Application No. 60/338,692, filed Dec. 11,2001. Application Ser. No. 10/225,067 is also a continuation-in-part of application Ser. No. 09/837,944, filed Apr. 18, 2001, abandoned, and also a continuation-in-part of application Ser. No. 10/171,468, filed Jun. 14, 2002, abandoned. The contents of application Ser. Nos. 09/837,944, 60/310,847, 09/934,455, 60/336,049, 60/338,692, 10/171,468, 10/225,066, 10/225,067, and 10/225,068 are hereby incorporated by reference in their entirety.

The claimed invention, in the field of functional genomics and the characterization of plant genes for the improvement of plants, was made by or on behalf of Mendel Biotechnology, Inc. and Monsanto Company as a result of activities undertaken within the scope of a joint research agreement, said agreement having been in effect on or before the date the claimed invention was made.

TECHNICAL FIELD

This invention relates to the field of plant biology. More particularly, the present invention pertains to compositions and methods for modifying a plant phenotypically.

BACKGROUND OF THE INVENTION

A plant's traits, such as its biochemical, developmental, or phenotypic characteristics, may be controlled through a number of cellular processes. One important way to manipulate that control is through transcription factors—proteins that influence the expression of a particular gene or sets of genes. Transformed and transgenic plants that comprise cells having altered levels of at least one selected transcription factor, for example, possess advantageous or desirable traits. Strategies for manipulating traits by altering a plant cell's transcription factor content can therefore result in plants and crops with new and/or improved commercially valuable properties.

Transcription factors can modulate gene expression, either increasing or decreasing (inducing or repressing) the rate of transcription. This modulation results in differential levels of gene expression at various developmental stages, in different tissues and cell types, and in response to different exogenous (e.g., environmental) and endogenous stimuli throughout the life cycle of the organism.

Because transcription factors are key controlling elements of biological pathways, alteringthe expression levels of one or more transcription factors can change entire biological pathways in an organism. For example, manipulation of the levels of selected transcription factors may result in increased expression of economically useful proteins or biomolecules in plants or improvement in other agriculturally relevant characteristics. Conversely, blocked or reduced expression of a transcription factor may reduce biosynthesis of unwanted compounds or remove an undesirable trait. Therefore, manipulating transcription factor levels in a plant offers tremendous potential in agricultural biotechnology for modifying a plant's traits. A number of the agriculturally relevant characteristics of plants, and desirable traits that may be imbued by gene expression are listed below.

Useful Plant Traits

Category: Abiotic Stress; Desired Trait: Chilling Tolerance

The term “chilling sensitivity” has been used to describe many types of physiological damage produced at low, but above freezing, temperatures. Most crops of tropical origins such as soybean, rice, maize and cotton are easily damaged by chilling. Typical chilling damage includes wilting, necrosis, chlorosis or leakage of ions from cell membranes. The underlying mechanisms of chilling sensitivity are not completely understood yet, but probably involve the level of membrane saturation and other physiological deficiencies. For example, photoinhibition of photosynthesis (disruption of photosynthesis due to high light intensities) often occurs under clear atmospheric conditions subsequent to cold late summer/autumn nights. By some estimates, chilling accounts for monetary losses in the United States (US) second only to drought and flooding. For example, chilling may lead to yield losses and lower product quality through the delayed ripening of maize. Another consequence of poor growth is the rather poor ground cover of maize fields in spring, often resulting in soil erosion, increased occurrence of weeds, and reduced uptake of nutrients. A retarded uptake of mineral nitrogen could also lead to increased losses of nitrate into the ground water.

Category: Abiotic Stress; Desired Trait: Freezing Tolerance.

Freezing is a major environmental stress that limits where crops can be grown and reduces yields considerably, depending on the weather in a particular growing season. In addition to exceptionally stressful years that cause measurable losses of billions of dollars, less extreme stress almost certainly causes smaller yield reductions over larger areas to produce yield reductions of similar dollar value every year. For instance, in the US, the 1995 early fall frosts are estimated to have caused losses of over one billion dollars to corn and soybeans. The spring of 1998 saw an estimated $200 M of damages to Georgia alone, in the peach, blueberry and strawberry industries. The occasional freezes in Florida have shifted the citrus belt further south due to $100 M or more losses. California sustained $650 M of damage in 1998 to the citrus crop due to a winter freeze. In addition, certain crops such as Eucalyptus, which has the very favorable properties of rapid growth and good wood quality for pulping, are not able to grow in the southeastern states due to occasional freezes.

Inherent winter hardiness of the crop determines in which agricultural areas it can survive the winter. For example, for wheat, the northern central portion of the US has winters that are too cold for good winter wheat crops. approximately 20% of the US wheat crop is spring wheat, with a market value of $2 billion. Areas growing spring wheat could benefit by growing winter wheat that had increased winter hardiness. Assuming a 25% yield increase when growing winter wheat, this would create $500 M of increased value. Additionally, the existing winter wheat is severely stressed by freezing conditions and should have improved yields with increased tolerance to these stresses. An estimate of the yield benefit of these traits is 10% of the $4.4 billion winter wheat crop in the US or $444 M of yield increase, as well as better survival in extreme freezing conditions that occur periodically.

Thus plants more resistant to freezing, both midwinter freezing and sudden freezes, would protect a farmers' investment, improve yield and quality, and allow some geographies to grow more profitable and productive crops. Additionally, winter crops such as canola, wheat and barley have 25% to 50% yield increases relative to spring planted varieties of the same crops. This yield increase is due to the “head start” the fall planted crop has over the spring planted crop and its reaching maturity earlier while the temperatures, soil moisture and lack of pathogens provide more favorable conditions.

Category: Abiotic Stress; Desired Trait: Salt Tolerance.

One in five hectares of irrigated land is damaged by salt, an important historical factor in the decline of ancient agrarian societies. This condition is only expected to worsen, further reducing the availability of arable land and crop production, since none of the top five food crops—wheat, corn, rice, potatoes, and soybean—can tolerate excessive salt.

Detrimental effects of salt on plants are a consequence of both water deficit resulting in osmotic stress (similar to drought stress) and the effects of excess sodium ions on critical biochemical processes. As with freezing and drought, high saline causes water deficit; the presence of high salt makes it difficult for plant roots to extract water from their environment (Buchanan et al. (2000) in Biochemistry and Molecular Biology of Plants, American Society of Plant Physiologists, Rockville, Md.). Soil salinity is thus one of the more important variables that determines where a plant may thrive. In many parts of the world, sizable land areas are uncultivable due to naturally high soil salinity. To compound the problem, salination of soils that are used for agricultural production is a significant and increasing problem in regions that rely heavily on agriculture. The latter is compounded by over-utilization, over-fertilization and water shortage, typically caused by climatic change and the demands of increasing population. Salt tolerance is of particular importance early in a plant's lifecycle, since evaporation from the soil surface causes upward water movement, and salt accumulates in the upper soil layer where the seeds are placed. Thus, germination normally takes place at a salt concentration much higher than the mean salt level in the whole soil profile.

Category: Abiotic Stress; Desired Trait: Drought Tolerance.

While much of the weather that we experience is brief and short-lived, drought is a more gradual phenomenon, slowly taking hold of an area and tightening its grip with time. In severe cases, drought can last for many years, and can have devastating effects on agriculture and water supplies. With burgeoning population and chronic shortage of available fresh water, drought is not only the number one weather related problem in agriculture, it also ranks as one of the major natural disasters of all time, causing not only economic damage, but also loss of human lives. For example, losses from the US drought of 1988 exceeded $40 billion, exceeding the losses caused by Hurricane Andrew in 1992, the Mississippi River floods of 1993, and the San Francisco earthquake in 1989. In some areas of the world, the effects of drought can be far more severe. In the Horn of Africa the 1984-1985 drought led to a famine that killed 750,000 people.

Problems for plants caused by low water availability include mechanical stresses caused by the withdrawal of cellular water. Drought also causes plants to become more susceptible to various diseases (Simpson (1981). “The Value of Physiological Knowledge of Water Stress in Plants”, In Water Stress on Plants, (Simpson, G. M., ed.), Praeger, NY, pp. 235-265).

In addition to the many land regions of the world that are too arid for most if not all crop plants, overuse and over-utilization of available water is resulting in an increasing loss of agriculturally-usable land, a process which, in the extreme, results in desertification. The problem is further compounded by increasing salt accumulation in soils, as described above, which adds to the loss of available water in soils.

Category: Abiotic Stress; Desired Trait: Heat Tolerance.

Germination of many crops is very sensitive to temperature. A transcription factor that would enhance germination in hot conditions would be useful for crops that are planted late in the season or in hot climates.

Seedlings and mature plants that are exposed to excess heat may experience heat shock, which may arise in various organs, including leaves and particularly fruit, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and cytoskeleton, and impairs membrane function (Buchanan, supra).

Heat shock may result a decrease in overall protein synthesis, accompanied by expression of heat shock proteins. Heat shock proteins function as chaperones and are involved in refolding proteins denatured by heat.

Category: Abiotic Stress; Desired Trait: Tolerance to Low Nitrogen and Phosphorus.

The ability of all plants to remove nutrients from their environment is essential to survival. Thus, identification of genes that encode polypeptides with transcription factor activity may allow for the generation of transgenic plants that are better able to make use of available nutrients in nutrient-poor environments.

Among the most important macronutrients for plant growth that have the largest impact on crop yield are nitrogenous and phosphorus-containing compounds. Nitrogen- and phosphorus-containing fertilizers are used intensively in agriculture practices today. An increase in grain crop yields from 0.5 to 1.0 metric tons per hectare to 7 metric tons per hectare accompanied the use of commercial fixed nitrogen fertilizer in production farming (Vance (2001) Plant Physiol. 127: 390-397). Given current practices, in order to meet food production demands in years to come, considerable increases in the amount of nitrogen- and phosphorus-containing fertilizers will be required (Vance, supra).

Nitrogen is the most abundant element in the Earth's atmosphere yet it is one of the most limiting elements to plant growth due to its lack of availability in the soil. Plants obtain N from the soil from several sources including commercial fertilizers, manure and the mineralization of organic matter. The intensive use of N fertilizers in present agricultural practices is problematic, the energy intensive Haber-Bosch process makes N fertilizer and it is estimated that the US uses annually between 3-5% of the nation's natural gas for this process. In addition to the expense of N fertilizer production and the depletion of non-renewable resources, the use of N fertilizers has led to the eutrophication of freshwater ecosystems and the contamination of drinking water due to the runoff of excess fertilizer into ground water supplies.

Phosphorus is second only to N in its importance as a macronutrient for plant growth and to its impact on crop yield. Phosphorus (P) is extremely immobile and not readily available to roots in the soil and is therefore often growth limiting to plants. Inorganic phosphate (Pi) is a constituent of several important molecules required for energy transfer, metabolic regulation and protein activation (Marschner (1995) Mineral Nutrition of Higher Plants, 2nd ed., Academic Press, San Diego, Calif.). Plants have evolved several strategies to help cope with P and N deprivation that include metabolic as well as developmental adaptations. Most, if not all, of these strategies have components that are regulated at the level of transcription and therefore are amenable to manipulation by transcription factors. Metabolic adaptations include increasing the availability of P and N by increasing uptake from the soil though the induction of high affinity and low affinity transporters, and/or increasing its mobilization in the plant. Developmental adaptations include increases in primary and secondary roots, increases in root hair number and length, and associations with mycorrhizal fungi (Bates and Lynch (1996) Plant Cell Environ. 19: 529-538; Harrison (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 361-389).

Category: Biotic Stress; Desired Trait: Disease Resistance.

Disease management is a significant expense in crop production worldwide. According to EPA reports for 1996 and 1997, US farmers spend approximately $6 billion on fungicides annually. Despite this expenditure, according to a survey conducted by the food and agriculture organization, plant diseases still reduce worldwide crop productivity by 12% and in the United States alone, economic losses due to plant pathogens amounts to 9.1 billion dollars (FAO, 1993). Data from these reports and others demonstrate that despite the availability of chemical control only a small proportion of the losses due to disease can be prevented. Not only are fungicides and anti-bacterial treatments expensive to growers, but their widespread application poses both environmental and health risks. The use of plant biotechnology to engineer disease resistant crops has the potential to make a significant economic impact on agriculture and forestry industries in two ways: reducing the monetary and environmental expense of fungicide application and reducing both pre-harvest and post-harvest crop losses that occur now despite the use of costly disease management practices.

Fungal, bacterial, oomycete, viral, and nematode diseases of plants are ubiquitous and important problems, and often severely impact yield and quality of crop and other plants. A very few examples of diseases of plants include:

Powdery mildew, caused by the fungi Erysiphe, Sphaerotheca, Phyllactinia, Microsphaera, Podosphaera, or Uncinula, in, for example, wheat, bean, cucurbit, lettuce, pea, grape, tree fruit crops, as well as roses, phlox, lilacs, grasses, and Euonymus;

Fusarium-caused diseases such as Fusarium wilt in cucurbits, Fusarium head blight in barley and wheat, wilt and crown and root rot in tomatoes;

Sudden oak death, caused by the oomycete Phytophthora ramorum; this disease was first detected in 1995 in California tan oaks. The disease has since killed more than 100,000 tan oaks, coast live oaks, black oaks, and Shreve's oaks in coastal regions of northern California, and more recently in southwestern Oregon (Roach (2001) National Geographic News, Dec. 6, 2001);

Black Sigatoka, a fungal disease caused by Mycosphaerella species that attacks banana foliage, is spreading throughout the regions of the world that are responsible for producing most of the world's banana crop;

Eutypa dieback, caused by Eutypa lata, affects a number of crop plants, including vine grape. Eutypa dieback delays shoot emergence, and causes chlorosis, stunting, and tattering of leaves;

Pierce's disease, caused by the bacterium Xylella fastidiosa, precludes growth of grapes in the southeastern United States, and threatens the profitable wine grape industry in northern California. The bacterium clogs the vasculature of the grapevines, resulting in foliar scorching followed by slow death of the vines. There is no known treatment for Pierce's disease;

Bacterial Spot caused by the bacterium Xanthomonas campestris causes serious disease problems on tomatoes and peppers. It is a significant problem in the Florida tomato industry because it spreads rapidly, especially in warm periods where there is wind-driven rain. Under these conditions, there are no adequate control measures;

Diseases caused by viruses of the family Geminiviridae are a growing agricultural problem worldwide. Geminiviruses have caused severe crop losses in tomato, cassaya, and cotton. For instance, in the 1991-1992 growing season in Florida, geminiviruses caused $140 million in damages to the tomato crop (Moffat (1991) Science 286: 1835). Geminiviruses have the ability to recombine between strains to rapidly produce new virulent varieties. Therefore, there is a pressing need for broad-spectrum geminivirus control;

The soybean cyst nematode, Heterodera glycines, causes stunting and chlorosis of soybean plants, which results in yield losses or plant death from severe infestation. Annual losses in the United States have been estimated at $1.5 billion (University of Minnesota Extension Service).

The aforementioned pathogens represent a very small fraction of diverse species that seriously affect plant health and yield. For a more complete description of numerous plant diseases, see, for example, Vidhyasekaran (1997) Fungal Pathogenesis in Plants and Crops: Molecular Biology and Host Defense Mechanisms, Marcel Dekker, Monticello, N.Y.), or Agrios (1997) Plant Pathology, Academic Press, New York, N.Y.). Plants that are able to resist disease may produce significantly higher yields and improved food quality. It is thus of considerable importance to find genes that reduce or prevent disease.

Category: Light Response; Desired Trait: Reduced Shade Avoidance.

Shade avoidance describes the process in which plants grown in close proximity attempt to out-compete each other by increasing stem length at the expense of leaf, fruit and storage organ development. This is caused by the plant's response to far-red radiation reflected from leaves of neighboring plants, which is mediated by phytochrome photoreceptors. Close proximity to other plants, as is produced in high-density crop plantings, increases the relative proportion of far-red irradiation, and therefore induces the shade avoidance response. Shade avoidance adversely affects biomass and yield, particularly when leaves, fruits or other storage organs constitute the desired crop (see, for example, Smith (1982) Annu. Rev. Plant Physiol. 33: 481-518; Ballare et al. (1990) Science 247: 329-332; Smith (1995) Annu. Dev. Plant Physiol. Mol. Biol., 46: 289-315; and Schmitt et al. (1995), American Naturalist, 146: 937-953). Alteration of the shade avoidance response in tobacco through alteration of phytochrome levels has been shown to produce an increase in harvest index (leaf biomass/total biomass) at high planting density, which would result in higher yield (Robson et al. (1996) Nature Biotechnol. 14: 995-998).

Category: Flowering Time; Desired Trait: Altered Flowering Time and Flowering Control.

Timing of flowering has a significant impact on production of agricultural products. For example, varieties with different flowering responses to environmental cues are necessary to adapt crops to different production regions or systems. Such a range of varieties have been developed for many crops, including wheat, corn, soybean, and strawberry. Improved methods for alteration of flowering time will facilitate the development of new, geographically adapted varieties.

Breeding programs for the development of new varieties can be limited by the seed-to-seed cycle. Thus, breeding new varieties of plants with multi-year cycles (such as biennials, e.g. carrot, or fruit trees, such as citrus) can be very slow. With respect to breeding programs, there would be a significant advantage in having commercially valuable plants that exhibit controllable and modified periods to flowering (“flowering times”). For example, accelerated flowering would shorten crop and tree breeding programs.

Improved flowering control allows more than one planting and harvest of a crop to be made within a single season. Early flowering would also improve the time to harvest plants in which the flower portion of the plant constitutes the product (e.g., broccoli, cauliflower, and other edible flowers). In addition, chemical control of flowering through induction or inhibition of flowering in plants could provide a significant advantage to growers by inducing more uniform fruit production (e.g., in strawberry)

A sizable number of plants for which the vegetative portion of the plant forms the valuable crop tend to “bolt” dramatically (e.g., spinach, onions, lettuce), after which biomass production declines and product quality diminishes (e.g., through flowering-triggered senescence of vegetative parts). Delay or prevention of flowering may also reduce or preclude dissemination of pollen from transgenic plants.

Category: Growth Rate; Desired Trait: Modified Growth Rate.

For almost all commercial crops, it is desirable to use plants that establish more quickly, since seedlings and young plants are particularly susceptible to stress conditions such as salinity or disease. Since many weeds may outgrow young crops or out-compete them for nutrients, it would also be desirable to determine means for allowing young crop plants to out compete weed species. Increasing seedling growth rate (emergence) contributes to seedling vigor and allows for crops to be planted earlier in the season with less concern for losses due to environmental factors. Early planting helps add days to the critical grain-filling period and increases yield.

Providing means to speed up or slow down plant growth would also be desirable to ornamental horticulture. If such means be provided, slow growing plants may exhibit prolonged pollen-producing or fruiting period, thus improving fertilization or extending harvesting season.

Category: Growth Rate; Desired Trait: Modified Senescence and Cell Death.

Premature senescence, triggered by various plant stresses, can limit production of both leaf biomass and seed yield. Transcription factor genes that suppress premature senescence or cell death in response to stresses can provide means for increasing yield. Delay of normal developmental senescence could also enhance yield, particularly for those plants for which the vegetative part of the plant represents the commercial product (e.g., spinach, lettuce).

Although leaf senescence is thought to be an evolutionary adaptation to recycle nutrients, the ability to control senescence in an agricultural setting has significant value. For example, a delay in leaf senescence in some maize hybrids is associated with a significant increase in yields and a delay of a few days in the senescence of soybean plants can have a large impact on yield. In an experimental setting, tobacco plants engineered to inhibit leaf senescence had a longer photosynthetic lifespan, and produced a 50% increase in dry weight and seed yield (Gan and Amasino (1995) Science 270: 1986-1988). Delayed flower senescence may generate plants that retain their blossoms longer and this may be of potential interest to the ornamental horticulture industry, and delayed foliar and fruit senescence could improve post-harvest shelf-life of produce.

Further, programmed cell death plays a role in other plant responses, including the resistance response to disease, and some symptoms of diseases, for example, as caused by necrotrophic pathogens such as Botrytis cinerea and Sclerotinia sclerotiorum (Dickman et al. Proc. Natl. Acad. Sci., 98: 6957-6962). Localized senescence and/or cell death can be used by plants to contain the spread of harmful microorganisms. A specific localized cell death response, the “hypersensitive response”, is a component of race-specific disease resistance mediated by plant resistance genes. The hypersensitive response is thought to help limit pathogen growth and to initiate a signal transduction pathway that leads to the induction of systemic plant defenses.

Accelerated senescence may be a defense against obligate pathogens, such as powdery mildew, that rely on healthy plant tissue for nutrients. With regard to powdery mildew, Botrytis cinerea and Sclerotinia sclerotiorum and other pathogens, transcription factors that ameliorate cell death and/or damage may reduce the significant economic losses encountered, such as, for example, Botrytis cinerea in strawberry and grape.

Category: Growth Regulator; Desired Trait: Altered Sugar Sensing

Sugars are key regulatory molecules that affect diverse processes in higher plants including germination, growth, flowering, senescence, sugar metabolism and photosynthesis. Sucrose, for example, is the major transport form of photosynthate and its flux through cells has been shown to affect gene expression and alter storage compound accumulation in seeds (source-sink relationships). Glucose-specific hexose-sensing has also been described in plants and is implicated in cell division and repression of “famine” genes (photosynthetic or glyoxylate cycles).

Category: Morphology; Desired Trait: Altered Morphology

Trichomes are branched or unbranched epidermal outgrowths or hair structures on a plant. Trichomes produce a variety of secondary biochemicals such as diterpenes and waxes, the former being important as, for example, insect pheromones, and the latter as protectants against desiccation and herbivorous pests. Since diterpenes also have commercial value as flavors, aromas, pesticides and cosmetics, and potential value as anti-tumor agents and inflammation-mediating substances, they have been both products and the target of considerable research. In most cases where the metabolic pathways are impossible to engineer, increasing trichome density or size on leaves may be the only way to increase plant productivity. Thus, it would be advantageous to discover trichome-affecting transcription factor genes for the purpose of increasing trichome density, size, or type to produce plants that are better protected from insects or that yield higher amounts of secondary metabolites.

The ability to manipulate wax composition, amount, or distribution could modify plant tolerance to drought and low humidity or resistance to insects, as well as plant appearance. In particular, a possible application for a transcription factor gene that reduces wax production in sunflower seed coats would be to reduce fouling during seed oil processing. Antisense or co-suppression of transcription factors involved in wax biosynthesis in a tissue specific manner can be used to specifically alter wax composition, amount, or distribution in those plants and crops from which wax is either a valuable attribute or product or an undesirable constituent of plants.

Other morphological characteristics that may be desirable in plants include those of an ornamental nature. These include changes in seed color, overall color, leaf and flower shape, leaf color, leaf size, or glossiness of leaves. Plants that produce dark leaves may have benefits for human health; flavonoids, for example, have been used to inhibit tumor growth, prevent of bone loss, and prevention lipid oxidation in animals and humans. Plants in which leaf size is increased would likely provide greater biomass, which would be particularly valuable for crops in which the vegetative portion of the plant constitutes the product. Plants with glossy leaves generally produce greater epidermal wax, which, if it could be augmented, resulted in a pleasing appearance for many ornamentals, help prevent desiccation, and resist herbivorous insects and disease-causing agents. Changes in plant or plant part coloration, brought about by modifying, for example, anthocyanin levels, would provide novel morphological features.

In many instances, the seeds of a plant constitute a valuable crop. These include, for example, the seeds of many legumes, nuts and grains. The discovery of means for producing larger seed would provide significant value by bringing about an increase in crop yield.

Plants with altered inflorescence, including, for example, larger flowers or distinctive floral configurations, may have high value in the ornamental horticulture industry.

Modifications to flower structure may have advantageous or deleterious effects on fertility, and could be used, for example, to decrease fertility by the absence, reduction or screening of reproductive components. This could be a desirable trait, as it could be exploited to prevent or minimize the escape of the pollen of genetically modified organisms into the environment.

Manipulation of inflorescence branching patterns may also be used to influence yield and offer the potential for more effective harvesting techniques. For example, a “self pruning” mutation of tomato results in a determinate growth pattern and facilitates mechanical harvesting (Pnueli et al. (2001) Plant Cell 13(12): 2687-2702).

Alterations of apical dominance or plant architecture could create new plant varieties. Dwarf plants may be of potential interest to the ornamental horticulture industry.

Category: Seed Biochemistry; Desired Trait: Altered Seed Oil

The composition of seeds, particularly with respect to seed oil quantity and/or composition, is very important for the nutritional value and production of various food and feed products. Desirable improvements to oils include enhanced heat stability, improved nutritional quality through, for example, reducing the number of calories in seed, increasing the number of calories in animal feeds, or altering the ratio of saturated to unsaturated lipids comprising the oils.

Category: Seed Biochemistry; Desired Trait: Altered Seed Protein

As with seed oils, seed protein content and composition is very important for the nutritional value and production of various food and feed products. Altered protein content or concentration in seeds may be used to provide nutritional benefits, and may also prolong storage capacity, increase seed pest or disease resistance, or modify germination rates. Altered amino acid composition of seeds, through altered protein composition, is also a desired objective for nutritional improvement.

Category: Seed Biochemistry; Desired Trait: Altered Prenyl Lipids.

Prenyl lipids, including the tocopherols, play a role in anchoring proteins in membranes or membranous organelles. Tocopherols have both anti-oxidant and vitamin E activity. Modified tocopherol composition of plants may thus be useful in improving membrane integrity and function, which may mitigate abiotic stresses such as heat stress. Increasing the anti-oxidant and vitamin content of plants through increased tocopherol content can provide useful human health benefits.

Category: Leaf Biochemistry; Desired Trait: Altered Glucosinolate Levels

Increases or decreases in specific glucosinolates or total glucosinolate content can be desirable depending upon the particular application. For example: (i) glucosinolates are undesirable components of the oilseeds used in animal feed, since they produce toxic effects; low-glucosinolate varieties of canola have been developed to combat this problem; (ii) some glucosinolates have anti-cancer activity; thus, increasing the levels or composition of these compounds can be of use in production of nutraceuticals; and (iii) glucosinolates form part of a plant's natural defense against insects; modification of glucosinolate composition or quantity could therefore afford increased protection from herbivores. Furthermore, tissue specific promoters can be used in edible crops to ensure that these compounds accumulate specifically in particular tissues, such as the epidermis, which are not taken for human consumption.

Category: Leaf Biochemistry; Desired Trait: Flavonoid Production.

Expression of transcription factors that increase flavonoid production in plants, including anthocyanins and condensed tannins, may be used to alter pigment production for horticultural purposes, and possibly to increase stress resistance. Flavonoids have antimicrobial activity and could be used to engineer pathogen resistance. Several flavonoid compounds have human health promoting effects such as inhibition of tumor growth, prevention of bone loss and prevention of lipid oxidation. Increased levels of condensed tannins in forage legumes would provide agronomic benefits in ruminants by preventing pasture bloat by collapsing protein foams within the rumen. For a review on the utilities of flavonoids and their derivatives, see Dixon et al. (1999) Trends Plant Sci. 4: 394-400.

The present invention relates to methods and compositions for producing transgenic plants with modified traits, particularly traits that address the agricultural and food needs described in the above background information. These traits may provide significant value in that they allow the plant to thrive in hostile environments, where, for example, temperature, water and nutrient availability or salinity may limit or prevent growth of non-transgenic plants. The traits may also comprise desirable morphological alterations, larger or smaller size, disease and pest resistance, alterations in flowering time, light response, and others.

We have identified polynucleotides encoding transcription factors, developed numerous transgenic plants using these polynucleotides, and have analyzed the plants for a variety of important traits. In so doing, we have identified important polynucleotide and polypeptide sequences for producing commercially valuable plants and crops as well as the methods for making them and using them. Other aspects and embodiments of the invention are described below and can be derived from the teachings of this disclosure as a whole.

SUMMARY OF THE INVENTION

Transgenic plants and methods for producing transgenic plants are provided. The transgenic plants comprise a recombinant polynucleotide having a polynucleotide sequence, or a sequence that is complementary to this polynucleotide sequence, that encodes a transcription factor.

The polynucleotide sequences that encode the transcription factors are listed in the Sequence Listing and include any of any of SEQ ID NO: 2N−1, wherein N=1-229, SEQ ID NO: 459-466; 468-487; 491-500; 504; 506-511; 516-520; 523-524; 527; 529; 531-533; 538-539; 541-557; 560-568; 570-586; 595-596; 598-606; 610-620; 627-634; 640-664; 670-707; 714-719; 722-735; 740-741; 743-779; 808-823; 825-834; 838-850; 855-864; 868-889; 892-902; 908-909; 914-921; 924-925; 927-932; 935-942; 944-952; 961-965; 968-986; 989-993; 995-1010; 1012-1034; 1043-1063; 1074-1080; 1091-1104; 1111-1121; 1123-1128; 1134-1138; 1142-1156; 1159-1175; 1187-1190; 1192-1199; 1202-1220; 1249-1253; 1258-1262; 1264-1269; 1271-1287; 1292-1301; 1303-1309; 1315-1323; 1328-1337; 1340-1341; 1344-1361; 1365-1377; 1379-1390; 1393-1394; 1396-1398; 1419-1432; 1434-1452; 1455-1456; 1460-1465; 1468-1491; 1499; 1502; 1505-1521; 1523-1527; 1529-1532; 1536-1539; 1542-1562; 1567-1571; 1573-1582; 1587-1592; 1595-1620; 1625-1644; 1647-1654; 1659-1669; 1671-1673; 1675-1680; 1682-1686; 1688-1700; 1706-1709; 1714-1726; 1728-1734; 1738-1742; 1744-1753; 1757-1760; 1763-1764; 1766-1768; 1770-1780; 1782-1784; 1786-1789; 1791-1804; 1806-1812; 1814-1837; 1847-1856; 1858-1862; 1864-1873; 1876-1882; 1885-1896; 1902-1910; 1913-1916; 1921-1928; 1931-1936; 1940-1941; 1944-1946, or SEQ ID NO: 2N−1, wherein N=974-1101.

The transcription factors are comprised of polypeptide sequences listed in the Sequence Listing and include any of SEQ ID NO: 2N, wherein N=1-229, SEQ ID NO: 467; 488-490; 501-503; 505; 512-515; 521-522; 525-526; 528; 530; 534-537; 540; 558-559; 569; 587-594; 597; 607-609; 621-626; 635-639; 665-669; 708-713; 720-721; 736-739; 742; 780-807; 824; 835-837; 851-854; 865-867; 890-891; 903-907; 910-913; 922-923; 926; 933-934; 943; 953-960; 966-967; 987-988; 994; 1011; 1035-1042; 1064-1073; 1081-1090; 1105-1110; 1122; 1129-1133; 1139-1141; 1157-1158; 1176-1186; 1191; 1200-1201; 1221-1248; 1254-1257; 1263; 1270; 1288-1291; 1302; 1310-1314; 1324-1327; 1338-1339; 1342-1343; 1362-1364; 1378; 1391-1392; 1395; 1399-1418; 1433; 1453-1454; 1457-1459; 1466-1467; 1492-1498; 1500-1501; 1503-1504; 1522; 1528; 1533-1535; 1540-1541; 1563-1566; 1572; 1583-1586; 1593-1594; 1621-1624; 1645-1646; 1655-1658; 1670; 1674; 1681; 1687; 1701-1705; 1710-1713; 1727; 1735-1737; 1743; 1754-1756; 1761-1762; 1765; 1769; 1781; 1785; 1790; 1805; 1813; 1838-1846; 1857; 1863; 1874-1875; 1883-1884; 1897-1901; 1911-1912; 1917-1920; 1929-1930; 1937-1939; 1942-1943; or SEQ ID NO: 2N, wherein N=974-1101.

The transgenic plant that comprises the recombinant polynucleotide has a polynucleotide sequence, or a sequence that is complementary to this polynucleotide sequence, selected from any of the following:

(a) a polynucleotide sequence that encodes one of the transcription factor polypeptide sequences of Paragraph 2 of this Summary; or

(b) a polynucleotide sequence that comprises one of the polynucleotide sequences of paragraph 3 of this Summary.

The transgenic plant may also comprise a polynucleotide sequence that is a variant of the sequences in (a) and (b) that encode a polypeptide and regulate transcription, including:

(c) a sequence variant of the polynucleotide sequences of (a) or (b);

(d) an allelic variant of the polynucleotide sequences of (a) or (b);

(e) a splice variant of the polynucleotide sequences of (a) or (b);

(f) an orthologous sequence of the polynucleotide sequences of (a) or (b);

(g) a paralogous sequence of the polynucleotide sequences of (a) or (b);

(h) a polynucleotide sequence encoding a polypeptide comprising a conserved domain that exhibits at least 70% sequence homology with the polypeptide of (a), and the polypeptide comprises a conserved domain of a transcription factor that regulates transcription; or

(i) a polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of one or more polynucleotides of (a) or (b), and the polynucleotide sequence encodes a polypeptide that regulates transcription.

A transcription factor sequence variant is one having at least 26% amino acid sequence similarity, or at least 40% amino acid sequence identity. A preferred transcription factor sequence variant is one having at least 50% amino acid sequence identity and a more preferred transcription factor sequence variant is one having at least 65% amino acid sequence identity to the transcription factor polypeptide sequences of paragraph 3 of this Summary, and that contains at least one functional or structural characteristic of the similar transcription factor polypeptide sequences. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.

The transcription factor polypeptides of the present invention include at least one conserved domain, and the portions of the polynucleotide sequences encoding the conserved domain generally exhibit at least 70% sequence identity with the aforementioned preferred polynucleotide sequences. In the case of zinc finger transcription factors, the percent identity across the conserved domain may be as low as 50%.

Various types of plants may be used to generate the transgenic plants, including soybean, wheat, corn, potato, cotton, rice, oilseed rape, sunflower, alfalfa, clover, sugarcane, turf, banana, blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon, mint and other labiates, rosaceous fruits, and vegetable brassicas.

The transgenic plant may be monocotyledonous, plant, and the polynucleotide sequences used to transform the transgenic plant may be derived from either a monocot or a dicot plant. Alternatively, the transgenic plant may be a dicotyledonous plant, and the polynucleotide sequences used to transform the transgenic plant may be derived from either a monocot or a dicot plant.

These transgenic plants will generally possess traits that are altered as compared to a control plant, such as a wild-type or non-transformed plant (i.e., the non-transformed plant does not comprise the recombinant polynucleotide), thus producing an phenotype that is altered when compared to the control, wild-type or non-transformed plant. These transgenic plants may also express an altered level of one or more genes associated with a plant trait as compared to the non-transformed plant. The encoded polypeptides in these transgenic plants will generally be expressed and regulate transcription of at least one gene; this gene will generally confer at least one altered trait, phenotype or expression level.

Any of the polynucleotide sequences listed in the Sequence Listing, their complements, and functional variants used to transform the transgenic plants of the present invention may further comprise regulatory elements. The regulatory elements, may comprise, for example, constitutive, inducible, or tissue-specific promoters operably linked to a polynucleotide sequence.

Presently disclosed transcription factor sequences may be used to produce transformed plants with a variety of improved traits. An example of such an altered trait is enhanced tolerance to abiotic stress, such as salt tolerance, chilling conditions, and drought conditions. Salt and drought tolerance, both forms of osmotic stress, may be mediatedin part by increased root growth or increased root hairs relative to a non-transformed, control or wild-type plant. Tolerance to abiotic stresses such as salt, chilling and drought tolerance may confer a number of survival, quality and yield improvements, including improved seed germination and improved seedling vigor, plant survival, as well as improved yield, quality, and range.

Another example of an altered trait that may be conferred by transforming plants with the presently disclosed transcription factor sequences includes altered sugar sensing. Altered sugar sensing may also be used to confer improved seed germination and improved seedling vigor, as well as altered flowering, senescence, sugar metabolism and photosynthesis characteristics.

The invention also pertains to method to produce these transgenic plants.

The present invention also relates to a method of using transgenic plants transformed with the presently disclosed transcription factor sequences, their complements or their variants to grow a progeny plant by crossing the transgenic plant with either itself or another plant, selecting seed that develops as a result of the crossing; and then growing the progeny plant from the seed. The progeny plant will generally express mRNA that encodes a transcription factor: that is, a DNA-binding protein that binds to a DNA regulatory sequence and regulates gene expression, such as that of a plant trait gene. The mRNA will generally be expressed at a level greater than a non-transformed plant; and the progeny plant is characterized by a change in a plant trait compared to the non-transformed plant.

The present invention also pertains to an expression cassette. The expression cassette comprises at least two elements, including:

(1) a constitutive, inducible, or tissue-specific promoter; and

(2) a recombinant polynucleotide having a polynucleotide sequence, or a complementary polynucleotide sequence thereof, selected from the group consisting of a polynucleotide sequence encoding a (a) polypeptide sequence selected from the transcription factor sequences in the third paragraph of this Summary; or (b) a polynucleotide sequence selected from the transcription factor polynucleotides of second paragraph of this Summary, or (c) sequence variants such as allelic or splice variants of the polynucleotide sequences of (a) or (b), where the sequence variant encodes a polypeptide that regulates transcription. The polynucleotide sequence may also comprise an orthologous or paralogous sequence of the polynucleotide sequences of (a) or (b), with these sequences encoding a polypeptide that regulates transcription, a polynucleotide sequence that encoding a polypeptide having a conserved domain that exhibits 72% or greater sequence homology with the polypeptide of (a), where the polypeptide comprising the conserved domain regulates transcription, or a polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence of one or more polynucleotides of (a) or (b), where the latter polynucleotide sequence regulates transcription. In all of these cases, the recombinant polynucleotide is operably linked to the promoter of the expression cassette.

The invention also includes a host cell that comprises the expression cassette. The host cell may be a plant cell, such as, for example, a cell of a crop plant.

The invention also concerns a method for identifying a factor that is modulated by or interacts with a polypeptide of the third paragraph of this Summary. This method is conducted by:expressing the polypeptide in a plant; and then identifying at least one factor that is modulated by or interacts with the polypeptide.

The invention also pertains to a method for identifying at least one downstream polynucleotide sequence that is subject to a regulatory effect of any of the polypeptides of the third paragraph of this Summary. This method includes expressing any of the polypeptides of the third paragraph of this Summary in a plant cell; and then identifying resultant RNA or protein. The latter identification may be carried out with, for example, such methods that include Northern analysis, RT-PCR, microarray gene expression assays, reporter gene expression systems subtractive hybridization, differential display, representational differential analysis, or two-dimensional gel electrophoresis of one or more protein products.

The invention also provides a transgenic plant comprising a polynucleotide encoding a polypeptide with a conserved domain, wherein the conserved domain comprises consecutive amino acid residues Ser-Ser-Lys/Arg-Tyr/Phe-Gly-Val-Val-Pro-Gln-Pro-Asn-Gly-Arg-Typ-Gly-Ala-Gln-Ile-Tyr-Glu-Lys/Arg-His-Gln-Arg-Val-Trp-Leu-Gly-Thr-Phe-Xaa-Glu/Asp-Glu-Glu/Asp-Glu/Asp-Ala-Ala/Val-Arg-Ala/Ser-Tyr-Asp-Val/Ile-Ala/Val-Val/Ala-Xaa-Arg-Phe/Tyr-Arg-Arg/Gly-Arg-Asp-Ala-Val-Thr/Val-Asn-Phe-Lys/Arg of SEQ ID NO: 170, wherein Xaa is any amino acid residue. The invention still further provides a transgenic plant comprising a polynucleotide wherein the polynucleotide sequence is selected from the group consisting of SEQ ID NO: 169, 369, 1159 through 1175, 1949, and 2071. In another embodiment, the invention also provides a transgenic plant comprising a polynucleotide encoding a polypeptide, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 170, 370, 1176 through 1186, 1950, and 2072.

The invention also provides an expression cassette comprising a polynucleotide encoding a polypeptide with a conserved domain, wherein the conserved domain comprises consecutive amino acid residues Ser-Ser-Lys/Arg-Tyr/Phe-Gly-Val-Val-Pro-Gln-Pro-Asn-Gly-Arg-Typ-Gly-Ala-Gln-Ile-Tyr-Glu-Lys/Arg-His-Gln-Arg-Val-Trp-Leu-Gly-Thr-Phe-Xaa-Glu/Asp-Glu-Glu/Asp-Glu/Asp-Ala-Ala/Val-Arg-Ala/Ser-Tyr-Asp-Val/Ile-Ala/Val-Val/Ala-Xaa-Arg-Phe/Tyr-Arg-Arg/Gly-Arg-Asp-Ala-Val-Thr/Val-Asn-Phe-Lys/Arg of SEQ ID NO:170, wherein Xaa is any amino acid residue. The invention still further provides an expression cassette comprising a polynucleotide sequence is selected from the group consisting of SEQ ID NO: 169, 369, 1159 through 1175, 1949, and 2071. In another embodiment, the invention also provides an expression cassette comprising a polynucleotide encoding a polypeptide, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 170, 370, 1176 through 1186, 1950, and 2072.

The invention also provides a method for producing a modified plant having a polynucleotide encoding a polypeptide with a conserved domain, wherein the conserved domain comprises consecutive amino acid residues Ser-Ser-Lys/Arg-Tyr/Phe-Gly-Val-Val-Pro-Gln-Pro-Asn-Gly-Arg-Typ-Gly-Ala-Gln-Ile-Tyr-Glu-Lys/Arg-His-Gln-Arg-Val-Trp-Leu-Gly-Thr-Phe-Xaa-Glu/Asp-Glu-Glu/Asp-Glu/Asp-Ala-Ala/Val-Arg-Ala/Ser-Tyr-Asp-Val/Ile-Ala/Val-Val/Ala-Xaa-Arg-Phe/Tyr-Arg-Arg/Gly-Arg-Asp-Ala-Val-Thr/Val-Asn-Phe-Lys/Arg of SEQ ID NO: 170, wherein Xaa is any amino acid residue. The invention still further provides a method for producing a modified plant having a polynucleotide, wherein the polynucleotide sequence is selected from the group consisting of SEQ ID NO: 169, 369, 1159 through 1175, 1949, and 2071. In another embodiment, the invention also provides a method for producing a modified plant having a polynucleotide encoding a polypeptide, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 170, 370, 1176 through 1186, 1950, and 2072.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS

The Sequence Listing provides exemplary polynucleotide and polypeptide sequences of the invention. The traits associated with the use of the sequences are included in the Examples.

CD-ROM1 (Copy 1) is a read-only memory computer-readable compact disc and contains a copy of the Sequence Listing in ASCII text format. The Sequence Listing is named “MBI0047.ST25.txt” and is 6,233 kilobytes in size. The copies of the Sequence Listing on the CD-ROM disc are hereby incorporated by reference in their entirety.

CD-ROM2 (Copy 2) is an exact copy of CD-R1 (Copy 1).

CD-ROM3 contains a computer-readable format (CRF) copy of the Sequence Listing as a text (.txt) file.

FIG. 1 shows a conservative estimate of phylogenetic relationships among the orders of flowering plants (modified from Angiosperm Phylogeny Group (1998) Ann. Missouri Bot. Gard. 84: 1-49). Those plants with a single cotyledon (monocots) are a monophyletic clade nested within at least two major lineages of dicots; the eudicots are further divided into rosids and asterids. Arabidopsis is a rosid eudicot classified within the order Brassicales; rice is a member of the monocot order Poales. FIG. 1 was adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333.

FIG. 2 shows a phylogenic dendogram depicting phylogenetic relationships of higher plant taxa, including clades containing tomato and Arabidopsis; adapted from Ku et al. (2000) Proc. Natl. Acad. Sci. 97: 9121-9126; and Chase et al. (1993) Ann. Missouri Bot. Gard. 80: 528-580.

FIGS. 3A, and 3B show an alignment of G682 (SEQ ID NO: 148) and polynucleotide sequences that are paralogous and orthologous to G682. The alignment was produced using MACVECTOR software (Acceirys, Inc., San Diego, Calif.).

FIGS. 4A, 4B, 4C and 4D show an alignment of G867 (SEQ ID NO: 170) and polynucleotide sequences that are paralogous and orthologous to G867. The alignment was produced using MACVECTOR software (Accelrys, Inc.).

FIGS. 5A, 5B, 5C, 5D, 5E and 5F show an alignment of G912 (SEQ ID NO: 186) and polynucleotide sequences that are paralogous and orthologous to G912. The alignment was produced using MACVECTOR software (Accelrys, Inc.).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

In an important aspect, the present invention relates to polynucleotides and polypeptides, for example, for modifying phenotypes of plants. Throughout this disclosure, various information sources are referred to and/or are specifically incorporated. The information sources include scientific journal articles, patent documents, textbooks, and World Wide Web browser-inactive page addresses, for example. While the reference to these information sources clearly indicates that they can be used by one of skill in the art, each and every one of the information sources cited herein are specifically incorporated in their entirety, whether or not a specific mention of “incorporation by reference” is noted. The contents and teachings of each and every one of the information sources can be relied on and used to make and use embodiments of the invention.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” includes a plurality of such plants, and a reference to “a stress” is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.

The polynucleotide sequences of the invention encode polypeptides that are members of well-known transcription factor families, including plant transcription factor families, as disclosed in Tables 4-5. Generally, the transcription factors encoded by the present sequences are involved in cellular metabolism, cell differentiation and proliferation and the regulation of growth. Accordingly, one skilled in the art would recognize that by expressing the present sequences in a plant, one may change the expression of autologous genes or induce the expression of introduced genes. By affecting the expression of similar autologous sequences in a plant that have the biological activity of the present sequences, or by introducing the present sequences into a plant, one may alter a plant's phenotype to one with improved traits. The sequences of the invention may also be used to transform a plant and introduce desirable traits not found in the wild-type cultivar or strain. Plants may then be selected for those that produce the most desirable degree of over- or under-expression of target genes of interest and coincident trait improvement.

The sequences of the present invention may be from any species, particularly plant species, in a naturally occurring form or from any source whether natural, synthetic, semi-synthetic or recombinant. The sequences of the invention may also include fragments of the present amino acid sequences. In this context, a “fragment” refers to a fragment of a polypeptide sequence which is at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity of a transcription factor. Where “amino acid sequence” is recited to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

As one of ordinary skill in the art recognizes, transcription factors can be identified by the presence of a region or domain of structural similarity or identity to a specific consensus sequence or the presence of a specific consensus DNA-binding site or DNA-binding site motif (see, for example, Riechmann et al. (2000) Science 290: 2105-2110). The plant transcription factors may belong to one of the following transcription factor families: the AP2 (APETALA2) domain transcription factor family (Riechmann and Meyerowitz (1998) Biol. Chem. 379: 633-646); the MYB transcription factor family (ENBib; Martin and Paz-Ares (1997) Trends Genet. 13: 67-73); the MADS domain transcription factor family (Riechmann and Meyerowitz (1997) Biol. Chem. 378: 1079-1101); the WRKY protein family (Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244: 563-571); the ankyrin-repeat protein family (Zhang et al. (1992) Plant Cell 4: 1575-1588); the zinc finger protein (Z) family (Klug and Schwabe (1995) FASEB J. 9: 597-604); Takatsuji (1998) Cell. Mol. Life Sci. 54:582-596); the homeobox (HB) protein family (Buerglin (1994) in Guidebook to the Homeobox Genes, Duboule (ed.) Oxford University Press); the CAAT-element binding proteins (Forsburg and Guarente (1989) Genes Dev. 3: 1166-1178); the squamosa promoter binding proteins (SPB) (Klein et al. (1996) Mol. Gen. Genet. 1996 250: 7-16); the NAM protein family (Souer et al. (1996) Cell 85: 159-170); the IAA/AUX proteins (Abel et al. (1995) J Mol. Biol. 251: 533-549); the HLH/MYC protein family (Littlewood et al. (1994) Prot. Profile 1: 639-709); the DNA-binding protein (DBP) family (Tucker et al. (1994) EMBO J 13: 2994-3002); the bZIP family of transcription factors (Foster et al. (1994) FASEB J. 8: 192-200); the Box P-binding protein (the BPF-1) family (da Costa e Silva et al. (1993) Plant J. 4: 125-135); the high mobility group (HMG) family (Bustin and Reeves (1996) Prog. Nucl. Acids Res. Mol. Biol. 54: 35-100); the scarecrow (SCR) family (Di Laurenzio et al. (1996) Cell 86: 423-433); the GF14 family (Wu et al. (1997) Plant Physiol. 114: 1421-1431); the polycomb (PCOMB) family (Goodrich et al. (1997) Nature 386: 44-51); the teosinte branched (TEO) family (Luo et al. (1996) Nature 383: 794-799); the ABI3 family (Giraudat et al. (1992) Plant Cell 4: 1251-1261); the triple helix (TH) family (Dehesh et al. (1990) Science 250: 1397-1399); the EIL family (Chao et al. (1997) Cell 89: 1133-44); the AT-HOOK family (Reeves and Nissen (1990) J. Biol. Chem. 265: 8573-8582); the SIFA family (Zhou et al. (1995) Nucleic Acids Res. 23: 1165-1169); the bZIPT2 family (Lu and Ferl (1995) Plant Physiol. 109: 723); the YABBY family (Bowman et al. (1999) Development 126: 2387-96); the PAZ family (Bohmert et al. (1998) EMBO J 17: 170-80); a family of miscellaneous (MISC) transcription factors including the DPBF family (Kim et al. (1997) Plant J. 11: 1237-1251) and the SPF1 family (Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244: 563-571); the GARP family (Hall et al. (1998) Plant Cell 10: 925-936), the TUBBY family (Boggin et al (1999) Science 286: 2119-2125), the heat shock family (Wu (1995) Annu. Rev. Cell Dev. Biol. 11: 441-469), the ENBP family (Christiansen et al. (1996) Plant Mol. Biol. 32: 809-821), the RING-zinc family (Jensen et al. (1998) FEBS Letters 436: 283-287), the PDBP family (Janik et al. (1989) Virology 168: 320-329), the PCF family (Cubas et al. Plant J. (1999) 18: 215-22), the SRS(SHI-related) family (Fridborg et al. (1999) Plant Cell 11: 1019-1032), the CPP (cysteine-rich polycomb-like) family (Cvitanich et al. (2000) Proc. Natl. Acad. Sci. 97: 8163-8168), the ARF (auxin response factor) family (Ulmasov et al. (1999) Proc. Natl. Acad. Sci. 96: 5844-5849), the SWI/SNF family (Collingwood et al. (1999) J. Mol. Endocrinol. 23: 255-275), the ACBF family (Seguin et al. (1997) Plant Mol. Biol. 35: 281-291), PCGL (CG-1 like) family (da Costa e Silva et al. (1994) Plant Mol. Biol. 25: 921-924) the ARID family (Vazquez et al. (1999) Development 126: 733-742), the Jumonji family (Balciunas et al. (2000), Trends Biochem. Sci. 25: 274-276), the bZIP-NIN family (Schauser et al. (1999) Nature 402: 191-195), the E2F family (Kaelin et al. (1992) Cell 70: 351-364) and the GRF-like family (Knaap et al. (2000) Plant Physiol. 122: 695-704). As indicated by any part of the list above and as known in the art, transcription factors have been sometimes categorized by class, family, and sub-family according to their structural content and consensus DNA-binding site motif, for example. Many of the classes and many of the families and sub-families are listed here. However, the inclusion of one sub-family and not another, or the inclusion of one family and not another, does not mean that the invention does not encompass polynucleotides or polypeptides of a certain family or sub-family. The list provided here is merely an example of the types of transcription factors and the knowledge available concerning the consensus sequences and consensus DNA-binding site motifs that help define them as known to those of skill in the art (each of the references noted above are specifically incorporated herein by reference). A transcription factor may include, but is not limited to, any polypeptide that can activate or repress transcription of a single gene or a number of genes. This polypeptide group includes, but is not limited to, DNA-binding proteins, DNA-binding protein binding proteins, protein kinases, protein phosphatases, protein methyltransferases, GTP-binding proteins, and receptors, and the like.

In addition to methods for modifying a plant phenotype by employing one or more polynucleotides and polypeptides of the invention described herein, the polynucleotides and polypeptides of the invention have a variety of additional uses. These uses include their use in the recombinant production (i.e., expression) of proteins; as regulators of plant gene expression, as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural coding nucleic acids); as substrates for further reactions, e.g., mutation reactions, PCR reactions, or the like; as substrates for cloning e.g., including digestion or ligation reactions; and for identifying exogenous or endogenous modulators of the transcription factors. A “polynucleotide” is a nucleic acid molecule comprising a plurality of polymerized nucleotides, e.g., at least about 15 consecutive polymerized nucleotides, optionally at least about 30 consecutive nucleotides, at least about 50 consecutive nucleotides. A polynucleotide may be a nucleic acid, oligonucleotide, nucleotide, or any fragment thereof. In many instances, a polynucleotide comprises a nucleotide sequence encoding a polypeptide (or protein) or a domain or fragment thereof. Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5′ or 3′ untranslated regions, a reporter gene, a selectable marker, or the like. The polynucleotide can be single stranded or double stranded DNA or RNA. The polynucleotide optionally comprises modified bases or a modified backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a transcript (such as an mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA, or the like. The polynucleotide can be combined with carbohydrate, lipids, protein, or other materials to perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). The polynucleotide can comprise a sequence in either sense or antisense orientations. “Oligonucleotide” is substantially equivalent to the terms amplimer, primer, oligoiner, element, target, and probe and is preferably single stranded.

Definitions

A “recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide comprises a nucleotide sequence not found in nature, or the polynucleotide is in a context other than that in which it is naturally found, e.g., separated from nucleotide sequences with which it typically is in proximity in nature, or adjacent (or contiguous with) nucleotide sequences with which it typically is not in proximity. For example, the sequence at issue can be cloned into a vector, or otherwise recombined with one or more additional nucleic acid.

An “isolated polynucleotide” is a polynucleotide whether naturally occurring or recombinant, that is present outside the cell in which it is typically found in nature, whether purified or not. Optionally, an isolated polynucleotide is subject to one or more enrichment or purification procedures, e.g., cell lysis, extraction, centrifugation, precipitation, or the like.

A “polypeptide” is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. A transcription factor can regulate gene expression and may increase or decrease gene expression in a plant. Additionally, the polypeptide may comprise 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) a DNA-binding domain, or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.

A “recombinant polypeptide” is a polypeptide produced by translation of a recombinant polynucleotide. A “synthetic polypeptide” is a polypeptide created by consecutive polymerization of isolated amino acid residues using methods well known in the art. An “isolated polypeptide,” whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%. Such an enrichment is not the result of a natural response of a wild-type plant. Alternatively, or additionally, the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods herein.

“Identity” or “similarity” refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences, with identity being a more strict comparison. The phrases “percent identity” and “% identity” refer to the percentage of sequence similarity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. “Sequence similarity” refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

“Alignment” refers to a number of DNA or amino acid sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. Alignments such as those of FIG. 3, 4, or 5 may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art, such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, Calif.).

The terms “highly stringent” or “highly stringent condition” refer to conditions that permit hybridization of DNA strands whose sequences are highly complementary, wherein these same conditions exclude hybridization of significantly mismatched DNAs. Polynucleotide sequences capable of hybridizing under stringent conditions with the polynucleotides of the present invention may be, for example, variants of the disclosed polynucleotide sequences, including allelic or splice variants, or sequences that encode orthologs or paralogs of presently disclosed polypeptides. Nucleic acid hybridization methods are disclosed in detail by Kashima et al. (1985) Nature 313:402-404, and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (“Sambrook”); and by Haymes et al., “Nucleic Acid Hybridization: A Practical Approach”, IRL Press, Washington, D.C. (1985), which references are incorporated herein by reference.

In general, stringency is determined by the temperature, ionic strength, and concentration of denaturing agents (e.g., formamide) used in a hybridization and washing procedure (for a more detailed description of establishing and determining stringency, see below). The degree to which two nucleic acids hybridize under various conditions of stringency is correlated with the extent of their similarity. Thus, similar nucleic acid sequences from a variety of sources, such as within a plant's genome (as in the case of paralogs) or from another plant (as in the case of orthologs) that may perform similar functions can be isolated on the basis of their ability to hybridize with known transcription factor sequences. Numerous variations are possible in the conditions and means by which nucleic acid hybridization can be performed to isolate transcription factor sequences having similarity to transcription factor sequences known in the art and are not limited to those explicitly disclosed herein. Such an approach may be used to isolate polynucleotide sequences having various degrees of similarity with disclosed transcription factor sequences, such as, for example, transcription factors having 60% identity, or more preferably greater than about 70% identity, most preferably 72% or greater identity with disclosed transcription factors.

The term “equivalog” describes members of a set of homologous proteins that are conserved with respect to function since their last common ancestor. Related proteins are grouped into equivalog families, and otherwise into protein families with other hierarchically defined homology types. This definition is provided at the Institute for Genomic Research (TIGR) website, www.tigr.org; “Terms associated with TIGRFAMs”.

The term “variant”, as used herein, may refer to polynucleotides or polypeptides that differ from the presently disclosed polynucleotides or polypeptides, respectively, in sequence from each other, and as set forth below.

With regard to polynucleotide variants, differences between presently disclosed polynucleotides and their variants are limited so that the nucleotide sequences of the former and the latter are closely similar overall and, in many regions, identical. The degeneracy of the genetic code dictates that many different variant polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing. Due to this degeneracy, differences between presently disclosed polynucleotides and variant nucleotide sequences may be silent in any given region or over the entire length of the polypeptide (i.e., the amino acids encoded by the polynucleotide are the same, and the variant polynucleotide sequence thus encodes the same amino acid sequence in that region or entire length of the presently disclosed polynucleotide. Variant nucleotide sequences may encode different amino acid sequences, in which case such nucleotide differences will result in amino acid substitutions, additions, deletions, insertions, truncations or fusions with respect to the similar disclosed polynucleotide sequences. These variations result in polynucleotide variants encoding polypeptides that share at least one functional characteristic (i.e., a presently disclosed transcription factor and a variant will confer at least one of the same functions to a plant).

Within the scope of the invention is a variant of a nucleic acid listed in the Sequence Listing (except CBF polynucleotide sequences SEQ ID NOs: 1955, 1957, 1959, or 2203), that is, one having a sequence that differs from the one of the polynucleotide sequences in the Sequence Listing, or a complementary sequence, that encodes a functionally equivalent polypeptide (i.e., a polypeptide having some degree of equivalent or similar biological activity) but differs in sequence from the sequence in the Sequence Listing, due to degeneracy in the genetic code.

“Allelic variant” or “polynucleotide allelic variant” refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations may be “silent” or may encode polypeptides having altered amino acid sequences. “Allelic variant” and “polypeptide allelic variant” may also be used with respect to polypeptides, and in this case the terms refer to a polypeptide encoded by an allelic variant of a gene.

“Splice variant” or “polynucleotide splice variant” as used herein refers to alternative forms of RNA transcribed from a gene. Splice variation naturally occurs as a result of alternative sites being spliced within a single transcribed RNA molecule or between separately transcribed RNA molecules, and may result in several different forms of mRNA transcribed from the same gene. Thus, splice variants may encode polypeptides having different amino acid sequences, which, in the present context, will have at least one similar function in the organism (splice variation may also give rise to distinct polypeptides having different functions). “Splice variant” or “polypeptide splice variant” may also refer to a polypeptide encoded by a splice variant of a transcribed mRNA.

As used herein, “polynucleotide variants” may also refer to polynucleotide sequences that encode paralogs and orthologs of the presently disclosed polypeptide sequences. “Polypeptide variants” may refer to polypeptide sequences that are paralogs and orthologs of the presently disclosed polypeptide sequences.

Differences between presently disclosed polypeptides and polypeptide variants are limited so that the sequences of the former and the latter are closely similar overall and, in many regions, identical. Presently disclosed polypeptide sequences and similar polypeptide variants may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. These differences may produce silent changes and result in a functionally equivalent transcription factor. Thus, it will be readily appreciated by those of skill in the art, that any of a variety of polynucleotide sequences is capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. A polypeptide sequence variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties. Deliberate amino acid substitutions may thus be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the functional or biological activity of the transcription factor is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine. For more detail on conservative substitutions, see Table 2. More rarely, a variant may have “non-conservative” changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing functional or biological activity may be found using computer programs well known in the art, for example, DNASTAR software (see U.S. Pat. No. 5,840,544).

The term “plant” includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/stiuctures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae. (See for example, FIG. 1, adapted from Daly et al. (2001) Plant Physiol. 127: 1328-1333; FIG. 2, adapted from Ku et al. (2000) Proc. Natl. Acad. Sci. 97: 9121-9126; and see also Tudge, in The Variety of Life, Oxford University Press, New York, N.Y. (2000) pp. 547-606).

A “transgenic plant” refers to a plant that contains genetic material not found in a wild-type plant of the same species, variety or cultivar. The genetic material may include a transgene, an insertional mutagenesis event (such as by transposon or T-DNA insertional mutagenesis), an activation tagging sequence, a mutated sequence, a homologous recombination event or a sequence modified by chimeraplasty. Typically, the foreign genetic material has been introduced into the plant by human manipulation, but any method can be used as one of skill in the art recognizes.

A transgenic plant may contain an expression vector or cassette. The expression cassette typically comprises a polypeptide-encoding sequence operably linked (i.e., under regulatory control of) to appropriate inducible or constitutive regulatory sequences that allow for the expression of polypeptide. The expression cassette can be introduced into a plant by transformation or by breeding after transformation of a parent plant. A plant refers to a whole plant, including seedlings and mature plants, as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells or any other plant material, e.g., a plant explant, as well as to progeny thereof, and to in vitro systems that mimic biochemical or cellular components or processes in a cell.

“Fragment”, with respect to a polynucleotide, refers to a clone or any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A polynucleotide fragment” refers to any subsequence of a polynucleotide, typically, of at least about 9 consecutive nucleotides, preferably at least about 30 nucleotides, more preferably at least about 50 nucleotides, of any of the sequences provided herein. Exemplary polynucleotide fragments are the first sixty consecutive nucleotides of the transcription factor polynucleotides listed in the Sequence Listing. Exemplary fragments also include fragments that comprise a region that encodes a conserved domain of a transcription factor.

Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide. Fragments may have uses in that they may have antigenic potential. In some cases, the fragment or domain is a subsequence of the polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA-binding site or domain that binds to a DNA promoter region, an activation domain, or a domain for protein-protein interactions, and may initiate transcription. Fragments can vary in size from as few as 3 amino acids to the full length of the intact polypeptide, but are preferably at least about 30 amino acids in length and more preferably at least about 60 amino acids in length. Exemplary polypeptide fragments are the first twenty consecutive amino acids of a mammalian protein encoded by are the first twenty consecutive amino acids of the transcription factor polypeptides listed in the Sequence Listing.

Exemplary fragments also include fragments that comprise a conserved domain of a transcription factor. An example of such an exemplary fragment would include amino acid residues 59-124 of G867 (SEQ ID NO: 170), as noted in Table 5.

The invention also encompasses production of DNA sequences that encode transcription factors and transcription factor derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding transcription factors or any fragment thereof.

A “conserved domain” or “conserved region” as used herein refers to a region in heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences.

With respect to polynucleotides encoding presently disclosed transcription factors, a conserved region is preferably at least 10 base pairs (bp) in length.

A “conserved domain”, with respect to presently disclosed polypeptides refers to a domain within a transcription factor family that exhibits a higher degree of sequence homology, such as at least 26% sequence similarity, at least 16% sequence identity, preferably at least 40% sequence identity, preferably at least 65% sequence identity including conservative substitutions, and more preferably at least 80% sequence identity, and even more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% amino acid residue sequence identity of a polypeptide of consecutive amino acid residues. A fragment or domain can be referred to as outside a conserved domain, outside a consensus sequence, or outside a consensus DNA-binding site that is known to exist or that exists for a particular transcription factor class, family, or sub-family. In this case, the fragment or domain will not include the exact amino acids of a consensus sequence or consensus DNA-binding site of a transcription factor class, family or sub-family, or the exact amino acids of a particular transcription factor consensus sequence or consensus DNA-binding site. Furthermore, a particular fragment, region, or domain of a polypeptide, or a polynucleotide encoding a polypeptide, can be “outside a conserved domain” if all the amino acids of the fragment, region, or domain fall outside of a defined conserved domain(s) for a polypeptide or protein. Sequences having lesser degrees of identity but comparable biological activity are considered to be equivalents.

As one of ordinary skill in the art recognizes, conserved domains of transcription factors may be identified as regions or domains of identity to a specific consensus sequence (see, for example, Riechmann et al. (2000) supra). Thus, by using alignment methods well known in the art, the conserved domains of the plant transcription factors for each of the following may be determined: the AP2 (APETALA2) domain transcription factor family (Riechmann and Meyerowitz (1998) supra; the MYB transcription factor family (ENBib; Martin and Paz-Ares (1997) supra); the MADS domain transcription factor family (Riechmann and Meyerowitz (1997) supra); the WRKY protein family (Ishiguro and Nakamura (1994) supra); the ankyrin-repeat protein family (Zhang et al. (1992) supra); the zinc finger protein (Z) family (Klug and Schwabe (1995) supra; Takatsuji (1998) supra); the homeobox (HB) protein family (Buerglin (1994) supra); the CAAT-element binding proteins (Forsburg and Guarente (1989) supra); the squamosa promoter binding proteins (SPB) (Klein et al. (1996) supra); the NAM protein family (Souer et al. (1996) supra); the IAA/AUX proteins (Abel et al. (1995) supra); the HLH/MYC protein family (Littlewood et al. (1994) supra); the DNA-binding protein (DBP) family (Tucker et al. (1994) supra); the bZIP family of transcription factors (Foster et al. (1994) supra); the Box P-binding protein (the BPF-1) family (da Costa e Silva et al. (1993) supra); the high mobility group (HMG) family (Bustin and Reeves (1996) supra); the scarecrow (SCR) family (Di Laurenzio et al. (1996) supra); the GF14 family (Wu et al. (1997) supra); the polycomb (PCOMB) family (Goodrich et al. (1997) supra); the teosinte branched (TEO) family (Luo et al. (1996) supra); the ABI3 family (Giraudat et al. (1992) supra); the triple helix (TH) family (Dehesh et al. (1990) supra); the EIL family (Chao et al. (1997) Cell supra); the AT-HOOK family (Reeves and Nissen (1990 supra); the SIFA family (Zhou et al. (1995) supra); the bZIPT2 family (Lu and Ferl (1995) supra); the YABBY family (Bowman et al. (1999) supra); the PAZ family (Bohmert et al. (1998) supra); a family of miscellaneous (MISC) transcription factors including the DPBF family (Kim et al. (1997) supra) and the SPF1 family (Ishiguro and Nakamura (1994) supra); the GARP family (Hall et al. (1998) supra), the TUBBY family (Boggin et al. (1999) supra), the heat shock family (Wu (1995 supra), the ENBP family (Christiansen et al. (1996) supra), the RING-zinc family (Jensen et al. (1998) supra), the PDBP family (Janik et al. (1989) supra), the PCF family (Cubas et al. (1999) supra), the SRS(SHI-related) family (Fridborg et al. (1999) supra), the CPP (cysteine-rich polycomb-like) family (Cvitanich et al. (2000) supra), the ARF (auxin response factor) family (Ulmasov et al. (1999) supra), the SWI/SNF family (Collingwood et al. (1999) supra), the ACBF family (Seguin et al. (1997) supra), PCGL (CG-1 like) family (da Costa e Silva et al. (1994) supra) the ARID family (Vazquez et al. (1999) supra), the Jumonji family, (Balciunas et al. (2000) supra), the bZIP-NIN family (Schauser et al. (1999) supra), the E2F family Kaelin et al. (1992) supra) and the GRF-like family (Knaap et al (2000) supra).

The conserved domains for each of polypeptides of SEQ ID NO: 2N, wherein N=1-229, are listed in Table 5 as described in Example VII. Also, many of the polypeptides of Table 5 have conserved domains specifically indicated by start and stop sites. A comparison of the regions of the polypeptides in SEQ ID NO: 2N, wherein N=1-229, or of those in Table 5, allows one of skill in the art to identify conserved domain(s) for any of the polypeptides listed or referred to in this disclosure, including those in Tables 4-8.

As used herein, a “gene” is a functional unit of inheritance, and in physical terms is a particular segment or sequence of nucleotides along a molecule of DNA (or RNA, in the case of RNA viruses) involved in producing a functional RNA molecule, such as one used for a structural or regulatory role, or a polypeptide chain, such as one used for a structural or regulatory role (an example of the latter would be transcription regulation, as by a transcription factor polypeptide). Polypeptides may then be subjected to subsequent processing such as splicing and/or folding to obtain a functional polypeptide. A gene may be isolated, partially isolated, or be found with an organism's genome. By way of example, a transcription factor gene encodes a transcription factor polypeptide, which may be functional withor without additional processing to function as an initiator of transcription.

Operationally, genes may be defined by the cis-trans test, a genetic test that determines whether two mutations occur in the same gene and which may be used to determine the limits of the genetically active unit (Rieger et al. (1976) Glossary of Genetics and Cytogenetics: Classical and Molecular, 4th ed., Springer Verlag. Berlin). A gene generally includes regions preceding (“leaders”; upstream) and following (“trailers”; downstream) of the coding region. A gene may also include intervening, non-coded sequences, referred to as “introns”, which are located between individual coding segments, referred to as “exons”. Most genes have an identifiable associated promoter region, a regulatory sequence 5′ or upstream of the transcription initiation codon. The function of a gene may also be regulated by enhancers, operators, and other regulatory elements.

A “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g. by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transgenic plants, however.

“Trait modification” refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease in an observed trait (difference), at least a 5% difference, at least about a 10% difference, at least about a 20% difference, at least about a 30%, at least about a 50%, at least about a 70%, or at least about a 100%, or an even greater difference compared with a wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution of the trait in the plants compared with the distribution observed in wild-type plant.

The term “transcript profile” refers to the expression levels of a set of genes in a cell in a particular state, particularly by comparison with the expression levels of that same set of genes in a cell of the same type in a reference state. For example, the transcript profile of a particular transcription factor in a suspension cell is the expression levels of a set of genes in a cell overexpressing that transcription factor compared with the expression levels of that same set of genes in a suspension cell that has normal levels of that transcription factor. The transcript profile can be presented as a list of those genes whose expression level is significantly different between the two treatments, and the difference ratios. Differences and similarities between expression levels may also be evaluated and calculated using statistical and clustering methods.

“Wild type”, as used herein, refers to a cell, tissue or plant that has not been genetically modified to knock out or overexpress one or more of the presently disclosed transcription factors. Wild-type cells, tissue or plants may be used as controls to compare levels of expression and the extent and nature of trait modification with modified (e.g., transgenic) cells, tissue or plants in which transcription factor expression is altered or ectopically expressed by, for example, knocking out or overexpressing a gene.

“Ectopic expression” or “altered expression” in reference to a polynucleotide indicates that the pattern of expression in, e.g., a transgenic plant or plant tissue, is different from the expression pattern in a wild-type plant or a reference plant of the same species. The pattern of expression may also be compared with a reference expression pattern in a wild-type plant of the same species. For example, the polynucleotide or polypeptide is expressed in a cell or tissue type other than a cell or tissue type in which the sequence is expressed in the wild-type plant, or by expression at a time other than at the time the sequence is expressed in the wild-type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild-type plant. Altered expression may be achieved by, for example, transformation of a plant with an expression cassette having a constitutive or inducible promoter element associated with a transcription factor gene. The resulting expression pattern can thus constitutive or inducible, and be stable or transient. Altered or ectopic expression may also refer to altered expression patterns that are produced by lowering the levels of expression to below the detection level or completely abolishing expression by, for example, knocking out a gene's expression by disrupting expression or regulation of the gene with an insertion element.

In reference to a polypeptide, the term “ectopic expression or altered expression” further may relate to altered activity levels resulting from the interactions of the polypeptides with exogenous or endogenous modulators or from interactions with factors or as a result of the chemical modification of the polypeptides.

The term “overexpression” as used herein refers to a greater expression level of a gene in a plant, plant cell or plant tissue, compared to expression in a wild-type plant, cell or tissue, at any developmental or temporal stage for the gene. Overexpression can occur when, for example, the genes encoding one or more transcription factors are under the control of a strong expression signal, such as one of the promoters described herein (e.g., the cauliflower mosaic virus 35S transcription initiation region). Overexpression may occur throughout a plant or in specific tissues of the plant, depending on the promoter used, as described below.

Overexpression may take place in plant cells normally lacking expression of polypeptides functionally equivalent or identical to the present transcription factors. Overexpression may also occur in plant cells where endogenous expression of the present transcription factors or functionally equivalent molecules normally occurs, but such normal expression is at a lower level than in the organism or tissues of the overexpressor. Overexpression thus results in a greater than normal production, or “overproduction” of the transcription factor in the plant, cell or tissue.

The term “phase change” refers to a plant's progression from embryo to adult, and, by some definitions, the transition wherein flowering plants gain reproductive competency. It is believed that phase change occurs either after a certain number of cell divisions in the shoot apex of a developing plant, or when the shoot apex achieves a particular distance from the roots. Thus, altering the timing of phase changes may affect a plant's size, which, in turn, may affect yield and biomass.

Traits That May Be Modified in Overexpressing or Knock-out Plants

Trait modifications of particular interest include those to seed (such as embryo or endosperm), fruit, root, flower, leaf, stem, shoot, seedling or the like, including: enhanced tolerance to environmental conditions including freezing, chilling, heat, drought, water saturation, radiation and ozone; improved tolerance to microbial, fungal or viral diseases; improved tolerance to pest infestations, including insects, nematodes, mollicutes, parasitic higher plants or the like; decreased herbicide sensitivity; improved tolerance of heavy metals or enhanced ability to take up heavy metals; improved growth under poor photoconditions (e.g., low light and/or short day length), or changes in expression levels of genes of interest. Other phenotype that can be modified relate to the production of plant metabolites, such as variations in the production of taxol, tocopherol, tocotrienol, sterols, phytosterols, vitamins, wax monomers, anti-oxidants, amino acids, lignins, cellulose, tannins, prenyllipids (such as chlorophylls and carotenoids), glucosinolates, and terpenoids, enhanced or compositionally altered protein or oil production (especially in seeds), or modified sugar (insoluble or soluble) and/or starch composition. Physical plant characteristics that can be modified include cell development (such as the number of trichomes), fruit and seed size and number, yields of plant parts such as stems, leaves, inflorescences, and roots, the stability of the seeds during storage, characteristics of the seed pod (e.g., susceptibility to shattering), root hair length and quantity, internode distances, or the quality of seed coat. Plant growth characteristics that can be modified include growth rate, germination rate of seeds, vigor of plants and seedlings, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, rate of nitrogen uptake, osmotic sensitivity to soluble sugar concentrations, biomass or transpiration characteristics, as well as plant architecture characteristics such as apical dominance, branching patterns, number of organs, organ identity, organ shape or size.

Transcription Factors Modify Expression of Endogenous Genes

Expression of genes that encode transcription factors that modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997) Genes and Development 11: 3194-3205, and Peng et al. (1999) Nature 400: 256-261. In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001) Plant Cell 13: 1791-1802; Nandi et al. (2000, Curr. Biol. 10: 215-218; Coupland (1995) Nature 377: 482-483; and Weigel and Nilsson (1995) Nature 377: 482-500.

In another example, Mandel et al. (1992) Cell 71-133-143 and Suzuki et al. (2001) Plant J. 28: 409-418, teach that a transcription factor expressed in another plant species elicits the same or very similar phenotypic response of the endogenous sequence, as often predicted in earlier studies of Arabidopsis transcription factors in Arabidopsis (see Mandel et al. (1992) supra; Suzuki et al. (2001) supra).

Other examples include Müller et al. (2001) Plant J. 28: 169-179; Kim et al. (2001) Plant J. 25: 247-259; Kyozuka and Shimamoto (2002) Plant Cell Physiol. 43: 130-135; Boss and Thomas (2002) Nature 416: 847-850; He et al. (2000) Transgenic Res. 9: 223-227; and Robson et al. (2001) Plant J. 28: 619-631.

In yet another example, Gilmour et al. (1998) Plant J. 16: 433-442, teach an Arabidopsis AP2 transcription factor, CBF1 (SEQ ID NO: 1956), which, when overexpressed in transgenic plants, increases plant freezing tolerance. Jaglo et al. (2001) Plant Physiol. 127: 910-917, further identified sequences in Brassica napus which encode CBF-like genes and that transcripts for these genes accumulated rapidly in response to low temperature. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat, as well as in tomato. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved consecutive amino acid residues, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. (See Jaglo et al. supra).

Gao et al. (2002) Plant Molec. Biol. 49: 459-471) have recently described four CBF transcription factors from Brassica napus: BNCBFs 5, 7, 16 and 17. They note that the first three CBFs (GenBank Accession Numbers AAM18958, AAM18959, and AAM18960, respectively) are very similar to Arabidopsis CBF1, whereas BNCBF17 (GenBank Accession Number AAM 18961) is similar but contains two extra regions of 16 and 21 amino acids in its acidic activation domain. All four B. napus CBFs accumulate in leaves of the plants after cold-treatment, and BNCBFs 5, 7, 16 accumulated after salt stress treatment. The authors concluded that these BNCBFs likely function in low-temperature responses in B. napus.

In a functional study of CBF genes, Hsieh et al. ((2002) Plant Physiol. 129: 1086-1094) found that heterologous expression of Arabidopsis CBF1 in tomato plants confers increased tolerance to chilling and considerable tolerance to oxidative stress, which suggested to the authors that ectopic Arabidopsis CBF1 expression may induce several tomato stress responsive genes to protect the plants.

Polypeptides and Polynucleotides of the Invention

The present invention provides, among other things, transcription factors (TFs), and transcription factor homolog polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides, or novel sequence variant polypeptides or polynucleotides encoding novel variants of transcription factors derived from the specific sequences provided here. These polypeptides and polynucleotides may be employed to modify a plant's characteristics.

Exemplary polynucleotides encoding the polypeptides of the invention were identified in the Arabidopsis thaliana GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. In addition, further exemplary polynucleotides encoding the polypeptides of the invention were identified in the plant GenBank database using publicly available sequence analysis programs and parameters. Sequences initially identified were then further characterized to identify sequences comprising specified sequence strings corresponding to sequence motifs present in families of known transcription factors. Polynucleotide sequences meeting such criteria were confirmed as transcription factors.

Additional polynucleotides of the invention were identified by screening Arabidopsis thaliana and/or other plant cDNA libraries with probes corresponding to known transcription factors under low stringency hybridization conditions. Additional sequences, including full length coding sequences were subsequently recovered by the rapid amplification of cDNA ends (RACE) procedure, using a commercially available kit according to the manufacturer's instructions. Where necessary, multiple rounds of RACE are performed to isolate 5′ and 3′ ends. The full-length cDNA was then recovered by a routine end-to-end polymerase chain reaction (PCR) using primers specific to the isolated 5′ and 3′ ends. Exemplary sequences are provided in the Sequence Listing.

The polynucleotides of the invention can be or were ectopically expressed in overexpressor or knockout plants and the changes in the characteristic(s) or trait(s) of the plants observed. Therefore, the polynucleotides and polypeptides can be employed to improve the characteristics of plants.

The polynucleotides of the invention can be or were ectopically expressed in overexpressor plant cells and the changes in the expression levels of a number of genes, polynucleotides, and/or proteins of the plant cells observed. Therefore, the polynucleotides and polypeptides can be employed to change expression levels of a genes, polynucleotides, and/or proteins of plants.

Producing Polypeptides

The polynucleotides of the invention include sequences that encode transcription factors and transcription factor homolog polypeptides and sequences complementary thereto, as well as unique fragments of coding sequence, or sequence complementary thereto. Such polynucleotides can be, e.g., DNA or RNA, e.g., mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides are either double-stranded or single-stranded, and include either, or both sense (i.e., coding) sequences and antisense (i.e., non-coding, complementary) sequences. The polynucleotides include the coding sequence of a transcription factor, or transcription factor homolog polypeptide, in isolation, in combination with additional coding sequences (e.g., a purification tag, a localization signal, as a fusion-protein, as a pre-protein, or the like), in combination with non-coding sequences (e.g., introns or inteins, regulatory elements such as promoters, enhancers, terminators, and the like), and/or in a vector or host environment in which the polynucleotide encoding a transcription factor or transcription factor homolog polypeptide is an endogenous or exogenous gene.

A variety of methods exist for producing the polynucleotides of the invention. Procedures for identifying and isolating DNA clones are well known to those of skill in the art, and are described in, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152 Academic Press, Inc., San Diego, Calif. (“Berger”); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology, Ausubel et al. eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2000) (“Ausubel”).

Alternatively, polynucleotides of the invention, can be produced by a variety of in vitro amplification methods adapted to the present invention by appropriate selection of specific or degenerate primers. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Qbeta-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), e.g., for the production of the homologous nucleic acids of the invention are found in Berger (supra), Sambrook (supra), and Ausubel (supra), as well as Mullis et al. (1987) PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis). Improved methods for cloning in vitro amplified nucleic acids are described in Wallace et al. U.S. Pat. No. 5,426,039. Improved methods for amplifying large nucleic acids by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the references cited therein, in which PCR amplicons of up to 40 kb are generated. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger, all supra.

Alternatively, polynucleotides and oligonucleotides of the invention can be assembled from fragments produced by solid-phase synthesis methods. Typically, fragments of up to approximately 100 bases are individually synthesized and then enzymatically or chemically ligated to produce a desired sequence, e.g., a polynucleotide encoding all or part of a transcription factor. For example, chemical synthesis using the phosphoramidite method is described, e.g., by Beaucage et al. (1981) Tetrahedron Letters 22: 1859-1869; and Matthes et al. (1984) EMBO J. 3: 801-805. According to such methods, oligonucleotides are synthesized, purified, annealed to their complementary strand, ligated and then optionally cloned into suitable vectors. And if so desired, the polynucleotides and polypeptides of the invention can be custom ordered from any of a number of commercial suppliers.

Homologous Sequences

Sequences homologous, i.e., that share significant sequence identity or similarity, to those provided in the Sequence Listing (except CBF sequences SEQ ID NOs: 1955-1960), derived from Arabidopsis thaliana or from other plants of choice, are also an aspect of the invention. Homologous sequences can be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn (maize), potato, cotton, rice, rape, oilseed rape (including canola), sunflower, alfalfa, clover, sugarcane, and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet corn, tobacco, tomato, tomatillo, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, Brussels sprouts, and kohlrabi). Other crops, including fruits and vegetables, whose phenotype can be changed and which comprise homologous sequences include barley; rye; millet; sorghum; currant; avocado; citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries; nuts such as the walnut and peanut; endive; leek; roots such as arrowroot, beet, cassaya, turnip, radish, yam, and sweet potato; and beans. The homologous sequences may also be derived from woody species, such pine, poplar and eucalyptus, or mint or other labiates. In addition, homologous sequences may be derived from plants that are evolutionarily-related to crop plants, but which may not have yet been used as crop plants. Examples include deadly nightshade (Atropa belladona), related to tomato; jimson weed (Datura strommium), related to peyote; and teosinte (Zea species), related to corn (maize).

Orthologs and Paralogs

Homologous sequences as described above can comprise orthologous or paralogous sequences. Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. Three general methods for defining orthologs and paralogs are described; an ortholog or paralog, including equivalogs, may be identified by one or more of the methods described below.

Orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.

Within a single plant species, gene duplication may cause two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs. A paralog is therefore a similar gene formed by duplication within the same species. Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) Methods Enzymol. 266: 383-402). Groups of similar genes can also be identified with pair-wise BLAST analysis (Feng and Doolittle (1987) J. Mol. Evol. 25: 351-360). For example, a clade of very similar MADS domain transcription factors from Arabidopsis all share a common function in flowering time (Ratcliffe et al. (2001) Plant Physiol. 126: 122-132), and a group of very similar AP2 domain transcription factors from Arabidopsis are involved in tolerance of plants to freezing (Gilmour et al. (1998) Plant J. 16: 433-442). Analysis of groups of similar genes with similar function that fall within one clade can yield sub-sequences that are particular to the clade. These sub-sequences, known as consensus sequences, can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount (2001), in Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., page 543.)

Speciation, the production of new species from a parental species, can also give rise to two or more genes with similar sequence and similar function. These genes, termed orthologs, often have an identical function within their host plants and are often interchangeable between species without losing function. Because plants have common ancestors, many genes in any plant species will have a corresponding orthologous gene in another plant species. Once a phylogenic tree for a gene family of one species has been constructed using a program such as CLUSTAL (Thompson et al. (1994) Nucleic Acids Res. 22: 4673-4680; Higgins et al. (1996) supra) potential orthologous sequences can be placed into the phylogenetic tree and their relationship to genes from the species of interest can be determined. Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.

Transcription factor gene sequences are conserved across diverse eukaryotic species lines (Goodrich et al. (1993) Cell 75: 519-530; Lin et al. (1991) Nature 353: 569-571; Sadowski et al. (1988) Nature 335: 563-564).et al. Plants are no exception to this observation; diverse plant species possess transcription factors that have similar sequences and functions.

Orthologous genes from different organisms have highly conserved functions, and very often essentially identical functions (Lee et al. (2002) Genome Res. 12: 493-502; Remm et al. (2001) J. Mol. Biol. 314: 1041-1052). Paralogous genes, which have diverged through gene duplication, may retain similar functions of the encoded proteins. In such cases, paralogs can be used interchangeably with respect to certain embodiments of the instant invention (for example, transgenic expression of a coding sequence). An example of such highly related paralogs is the CBF family, with three well-defined members in Arabidopsis and at least one ortholog in Brassica napus (SEQ ID NOs: 1956, 1958, 1960, or 2204, respectively), all of which control pathways involved in both freezing and drought stress (Gilmour et al. (1998) Plant J. 16: 433-442; Jaglo et al. (1998) Plant Physiol. 127: 910-917).

The following references represent a small sampling of the many studies that demonstrate that conserved transcription factor genes from diverse species are likely to function similarly (i.e., regulate similar target sequences and control the same traits), and that transcription factors may be transformed into diverse species to confer or improve traits.

(1) The Arabidopsis NPR1 gene regulates systemic acquired resistance (SAR); over-expression of NPR1 leads to enhanced resistance in Arabidopsis. When either Arabidopsis NPR1 or the rice NPR1 ortholog was overexpressed in rice (which, as a monocot, is diverse from Arabidopsis), challenge with the rice bacterial blight pathogen Xanthomonas oryzae pv. Oryzae, the transgenic plants displayed enhanced resistance (Chern et al. (2001) Plant J. 27: 101-113). NPR1 acts through activation of expression of transcription factor genes, such as TGA2 (Fan and Dong (2002) Plant Cell 14: 1377-1389).

(2) E2F genes are involved in transcription of plant genes for proliferating cell nuclear antigen (PCNA). Plant E2Fs share a high degree of similarity in amino acid sequence between monocots and dicots, and are even similar to the conserved domains of the animal E2Fs. Such conservation indicates a functional similarity between plant and animal E2Fs. E2F transcription factors that regulate meristem development act through common cis-elements, and regulate related (PCNA) genes (Kosugi and Ohashi, (2002) Plant J. 29: 45-59).

(3) The ABI5 gene (abscisic acid (ABA) insensitive 5) encodes a basic leucine zipper factor required for ABA response in the seed and vegetative tissues. Co-transformation experiments with ABI5 cDNA constructs in rice protoplasts resulted in specific transactivation of the ABA-inducible wheat, Arabidopsis, bean, and barley promoters. These results demonstrate that sequentially similar ABI5 transcription factors are key targets of a conserved ABA signaling pathway in diverse plants. (Gampala et al. (2001) J. Biol. Chem. 277: 1689-1694).

(4) Sequences of three Arabidopsis GAMYB-like genes were obtained on the basis of sequence similarity to GAMYB genes from barley, rice, and L. temulentum. These three Arabadopsis genes were determined to encode transcription factors (AtMYB33, AtMYB65, and AtMYB101) and could substitute for a barley GAMYB and control alpha-amylase expression (Gocal et al. (2001) Plant Physiol. 127: 1682-1693).

(5) The floral control gene LEAFY from Arabidopsis can dramatically accelerate flowering in numerous dictoyledonous plants. Constitutive expression of Arabidopsis LEAFY also caused early flowering in transgenic rice (a monocot), with a heading date that was 26-34 days earlier than that of wild-type plants. These observations indicate that floral regulatory genes from Arabidopsis are useful tools for heading date improvement in cereal crops (He et al. (2000) Transgenic Res. 9: 223-227).

(6) Bioactive gibberellins (GAs) are essential endogenous regulators of plant growth. GA signaling tends to be conserved across the plant kingdom. GA signaling is mediated via GAI, a nuclear member of the GRAS family of plant transcription factors. Arabidopsis GAI has been shown to function in rice to inhibit gibberellin response pathways (Fu et al. (2001) Plant Cell 13: 1791-1802).

(7) The Arabidopsis gene SUPERMAN (SUP), encodes a putative transcription factor that maintains the boundary between stamens and carpels. By over-expressing Arabidopsis SUP in rice, the effect of the gene's presence on whorl boundaries was shown to be conserved. This demonstrated that SUP is a conserved regulator of floral whorl boundaries and affects cell proliferation (Nandi et al. (2000) Curr. Biol. 10: 215-218).

(8) Maize, petunia and Arabidopsis myb transcription factors that regulate flavonoid biosynthesis are very genetically similar and affect the same trait in their native species, therefore sequence and function of these myb transcription factors correlate with each other in these diverse species (Borevitz et al. (2000) Plant Cell 12: 2383-2394).

(9) Wheat reduced height-1 (Rht-B1/Rht-D1) and maize dwarf-8 (d8) genes are orthologs of the Arabidopsis gibberellin insensitive (GAI) gene. Both of these genes have been used to produce dwarf grain varieties that have improved grain yield. These genes encode proteins that resemble nuclear transcription factors and contain an SH2-like domain, indicating that phosphotyrosine may participate in gibberellin signaling. Transgenic rice plants containing a mutant GAI allele from Arabidopsis have been shown to produce reduced responses to gibberellin and are dwarfed, indicating that mutant GAI orthologs could be used to increase yield in a wide range of crop species (Peng et al. (1999) Nature 400: 256-261).

Transcription factors that are homologous to the listed sequences will typically share, in at least one conserved domain, at least about 70% amino acid sequence identity, and with regard to zinc finger transcription factors, at least about 50% amino acid sequence identity. More closely related transcription factors can share at least about 70%, or about 75% or about 80% or about 90% or about 95% or about 98% or more sequence identity with the listed sequences, or with the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site, or with the listed sequences excluding one or all conserved domain. Factors that are most closely related to the listed sequences share, e.g., at least about 85%, about 90% or about 95% or more % sequence identity to the listed sequences, or to the listed sequences but excluding or outside a known consensus sequence or consensus DNA-binding site or outside one or all conserved domain. At the nucleotide level, the sequences will typically share at least about 40% nucleotide sequence identity, preferably at least about 50%, about 60%, about 70% or about 80% sequence identity, and more preferably about 85%, about 90%, about 95% or about 97% or more sequence identity to one or more of the listed sequences, or to a listed sequence but excluding or outside a known consensus sequence or consensus DNA-binding site, or outside one or all conserved domain. The degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein. Conserved domains within a transcription factor family may exhibit a higher degree of sequence homology, such as at least 65% amino acid sequence identity including conservative substitutions, and preferably at least 80% sequence identity, and more preferably at least 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 90%, or at least about 95%, or at least about 98% sequence identity. Transcription factors that are homologous to the listed sequences should share at least 30%, or at least about 60%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 95% amino acid sequence identity over the entire length of the polypeptide or the homolog.

Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc. Madison, Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, for example, the clustal method. (See, for example, Higgins and Sharp (1988) Gene 73: 237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. Other alignment algorithms or programs may be used, including FASTA, BLAST, or ENTREZ, FASTA and BLAST, and which may be used to calculate percent similarity. These are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with or without default settings. ENTREZ is available through the National Center for Biotechnology Information. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences (see U.S. Pat. No. 6,262,333).

Other techniques for alignment are described in Doolittle, R. F. (1996) Methods in Enzymology: Computer Methods for Macromolecular Sequence Analysis, vol. 266, Academic Press, Orlando, Fla., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments (see Shpaer (1997) Methods Mol. Biol. 70: 173-187). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.

The percentage similarity between two polypeptide sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between polynucleotide sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein (1990) Methods Enzymol. 183: 626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions (see US Patent Application No. 20010010913).

The percent identity between two conserved domains of a transcription factor DNA-binding domain consensus polypeptide sequence can be as low as 16%, as exemplified in the case of GATA1 family of eukaryotic Cys2/Cys2-type zinc finger transcription factors. The DNA-binding domain consensus polypeptide sequence of the GATA1 family is CX2CX17CX2C, where X is any amino acid residue. (See, for example, Takatsuji, supra.) Other examples of such conserved consensus polypeptide sequences with low overall percent sequence identity are well known to those of skill in the art.

Thus, the invention provides methods for identifying a sequence similar or paralogous or orthologous or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an internet or intranet) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

In addition, one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to search against a BLOCKS (Bairoch et al. (1997) Nucleic Acids Res. 25: 217-221), PFAM, and other databases which contain previously identified and annotated motifs, sequences and gene functions. Methods that search for primary sequence patterns with secondary structure gap penalties (Smith et al. (1992) Protein Engineering 5: 35-51) as well as algorithms such as Basic Local Alignment Search Tool (BLAST; Altschul (1993) J. Mol. Evol. 36: 290-300; Altschul et al. (1990) supra), BLOCKS (Henikoff and Henikoff (1991) Nucleic Acids Res. 19: 6565-6572), Hidden Markov Models (HMM; Eddy (1996) Curr. Opin. Str. Biol. 6: 361-365; Sonnhammer et al. (1997) Proteins 28: 405-420), and the like, can be used to manipulate and analyze polynucleotide and polypeptide sequences encoded by polynucleotides. These databases, algorithms and other methods are well known in the art and are described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York, N.Y., p 856-853).

Furthermore, methods using manual alignment of sequences similar or homologous to one or more polynucleotide sequences or one or more polypeptides encoded by the polynucleotide sequences may be used to identify regions of similarity and conserved domains. Such manual methods are well-known of those of skill in the art and can include, for example, comparisons of tertiary structure between a polypeptide sequence encoded by a polynucleotide which comprises a known function with a polypeptide sequence encoded by a polynucleotide sequence which has a function not yet determined. Such examples of tertiary structure may comprise predicted alpha helices, beta-sheets, amphipathic helices, leucine zipper motifs, zinc finger motifs, proline-rich regions, cysteine repeat motifs, and the like.

Orthologs and paralogs of presently disclosed transcription factors may be cloned using compositions provided by the present invention according to methods well known in the art. cDNAs can be cloned using mRNA from a plant cell or tissue that expresses one of the present transcription factors. Appropriate mRNA sources may be identified by interrogating Northern blots with probes designed from the present transcription factor sequences, after which a library is prepared from the mRNA obtained from a positive cell or tissue. Transcription factor-encoding cDNA is then isolated using, for example, PCR, using primers designed from a presently disclosed transcription factor gene sequence, or by probing with a partial or complete cDNA or with one or more sets of degenerate probes based on the disclosed sequences. The cDNA library may be used to transform plant cells. Expression of the cDNAs of interest is detected using, for example, methods disclosed herein such as microarrays, Northern blots, quantitative PCR, or any other technique for monitoring changes in expression. Genomic clones may be isolated using similar techniques to those.

Identifying Polynucleotides or Nucleic Acids by Hybridization

Polynucleotides homologous to the sequences illustrated in the Sequence Listing and tables can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc. present in both the hybridization and wash solutions and incubations (and number thereof, as described in more detail in the references cited above.

Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the transcription factor polynucleotides within the Sequence Listing (excluding CBF sequences SEQ ID NOs: 1955, 1957, 1959, or 2203), and fragments thereof under various conditions of stringency (See, for example, Wahl and Berger (1987) Methods Enzymol. 152: 399-407; and Kimmel (1987) Methods Enzymol. 152: 507-511). In addition to the nucleotide sequences listed in Tables 4 and 5, full length cDNA, orthologs, and paralogs of the present nucleotide sequences may be identified and isolated using well-known methods. The cDNA libraries orthologs, and paralogs of the present nucleotide sequences may be screened using hybridization methods to determine their utility as hybridization target or amplification probes.

With regard to hybridization, conditions that are highly stringent, and means for achieving them, are well known in the art. See, for example, Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual” (2nd ed., Cold Spring Harbor Laboratory); Berger and Kimmel, eds., (1987) “Guide to Molecular Cloning Techniques”, In Methods in Enzymology:152: 467-469; and Anderson and Young (1985) “Quantitative Filter Hybridisation.” In: Hames and Higgins, ed., Nucleic Acid Hybridisation, A Practical Approach. Oxford, IRL Press, 73-111.

Stability of DNA duplexes is affected by such factors as base composition, length, and degree of base pair mismatch. Hybridization conditions may be adjusted to allow DNAs of different sequence relatedness to hybridize. The melting temperature (Tm) is defined as the temperature when 50% of the duplex molecules have dissociated into their constituent single strands. The melting temperature of a perfectly matched duplex, where the hybridization buffer contains formamide as a denaturing agent, may be estimated by the following equation:
DNA-DNA: T m(° C.)=81.5+16.6(log [Na+])+0.41(% G+C)−0.62(% formamide)−500/L  (1)
DNA-RNA: T m(° C.)=79.8+18.5(log [Na+])+0.58(% G+C)+0.12(% G+C)2−0.5(% formamide)−820/L  (2)
RNA-RNA: T m(° C.)=79.8+18.5(log [Na+])+0.58(%G+C)+0.12(%G+C)2−0.35(% formamide)−820/L  (3)

where L is the length of the duplex formed, [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution, and % G+C is the percentage of (guanine+cytosine) bases in the hybrid. For imperfectly matched hybrids, approximately 1° C. is required to reduce the melting temperature for each 1-% mismatch.

Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson et al. (1985) supra). In addition, one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinyl-pyrrolidone, ficoll and Denhardt's solution. Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time. In some instances, conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.

Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms. The stringency can be adjusted either during the hybridization step or in the post-hybridization washes. Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency (as described by the formula above). As a general guidelines high stringency is typically performed at Tm−5oC to Tm−20oC, moderate stringency at Tm−20oC to Tm−35oC and low stringency at Tm−35oC to Tm−50oC for duplex >150 base pairs. Hybridization may be performed at low to moderate stringency (25-50oC below Tm), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at Tm−25oC for DNA-DNA duplex and Tm−15oC for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.

High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences. An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Conditions used for hybridization may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at hybridization temperatures between about 50° C. and about 70° C. More preferably, high stringency conditions are about 0.02 M sodium chloride, about 0.5% casein, about 0.02% SDS, about 0.001 M sodium citrate, at a temperature of about 50° C. Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire DNA molecule or selected portions, e.g., to a unique subsequence, of the DNA.

Stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate. Increasingly stringent conditions may be obtained with less than about 500 mM NaCl and 50 mM trisodium citrate, to even greater stringency with less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whereas high stringency hybridization may be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. with formamide present. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS) and ionic strength, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide. In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide. Useful variations on these conditions will be readily apparent to those skilled in the art.

The washing steps that follow hybridization may also vary in stringency; the post-hybridization wash steps primarily determine hybridization specificity, with the most critical factors being temperature and the ionic strength of the final wash solution. Wash stringency can be increased by decreasing salt concentration or by increasing temperature. Stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. For example, the wash conditions may be under conditions of 0.1×SSC to 2.0×SSC and 0.1% SDS at 50-65° C., with, for example, two steps of 10-30 min. One example of stringent wash conditions includes about 2.0×SSC, 0.1% SDS at 65° C. and washing twice, each wash step being about 30 min. A higher stringency wash is about 0.2×SSC, 0.1% SDS at 65° C. and washing twice for 30 min. A still higher stringency wash is about 0.1×SSC, 0.1% SDS at 65° C. and washing twice for 30 min. The temperature for the wash solutions will ordinarily be at least about 25° C., and for greater stringency at least about 42° C. Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3° C. to about 5° C., and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6° C. to about 9° C. For identification of less closely related homolog, wash steps may be performed at a lower temperature, e.g., 50° C.

An example of a low stringency wash step employs a solution and conditions of at least 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may be obtained at 42° C. in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Even higher stringency wash conditions are obtained at 65° C.-68° C. in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, US Patent Application No. 20010010913).

Stringency conditions can be selected such that an oligonucleotide that is perfectly complementary to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10× higher signal to noise ratio than the ratio for hybridization of the perfectly complementary oligonucleotide to a nucleic acid encoding a transcription factor known as of the filing date of the application. It may be desirable to select conditions for a particular assay such that a higher signal to noise ratio, that is, about 15× or more, is obtained. Accordingly, a subject nucleic acid will hybridize to a unique coding oligonucleotide with at least a 2× or greater signal to noise ratio as compared to hybridization of the coding oligonucleotide to a nucleic acid encoding known polypeptide. The particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a calorimetric label, a radioactive label, or the like. Labeled hybridization or PCR probes for detecting related polynucleotide sequences may be produced by oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Identifying Polynucleotides or Nucleic Acids with Expression Libraries

In addition to hybridization methods, transcription factor homolog polypeptides can be obtained by screening an expression library using antibodies specific for one or more transcription factors. With the provision herein of the disclosed transcription factor, and transcription factor homolog nucleic acid sequences, the encoded polypeptide(s) can be expressed and purified in a heterologous expression system (e.g., E. coli) and used to raise antibodies (monoclonal or polyclonal) specific for the polypeptide(s) in question. Antibodies can also be raised against synthetic peptides derived from transcription factor, or transcription factor homolog, amino acid sequences. Methods of raising antibodies are well known in the art and are described in Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone additional transcription factor homologs, using the methods described above. The selected cDNAs can be confirmed by sequencing and enzymatic activity.

Sequence Variations

It will readily be appreciated by those of skill in the art, that any of a variety of polynucleotide sequences are capable of encoding the transcription factors and transcription factor homolog polypeptides of the invention. Due to the degeneracy of the genetic code, many different polynucleotides can encode identical and/or substantially similar polypeptides in addition to those sequences illustrated in the Sequence Listing (except CBF polypeptide sequences SEQ ID NOs: 1956, 1958, 1960, or 2204). Nucleic acids having a sequence that differs from the sequences shown in the Sequence Listing, or complementary sequences, that encode functionally equivalent peptides (i.e., peptides having some degree of equivalent or similar biological activity) but differ in sequence from the sequence shown in the Sequence Listing due to degeneracy in the genetic code, are also within the scope of the invention.

Altered polynucleotide sequences encoding polypeptides include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide encoding a polypeptide with at least one functional characteristic of the instant polypeptides. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding the instant polypeptides, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding the instant polypeptides.

Allelic variant refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (i.e., no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. Splice variant refers to alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.

Those skilled in the art would recognize that, for example, G28, SEQ ID NO: 10, represents a single transcription factor; allelic variation and alternative splicing may be expected to occur. Allelic variants of SEQ ID NO: 9 can be cloned by probing cDNA or genomic libraries from different individual organisms according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 9, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 10. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the transcription factor are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individual organisms or tissues according to standard procedures known in the art (see U.S. Pat. No. 6,388,064).

Thus, in addition to the sequences set forth in the Sequence Listing (except CBF sequences), the invention also encompasses related nucleic acid molecules that include allelic or splice variants of SEQ ID NO: 2N−1, wherein N=1-229, SEQ ID NO: 459-466; 468-487; 491-500; 504; 506-511; 516-520; 523-524; 527; 529; 531-533; 538-539; 541-557; 560-568; 570-586; 595-596; 598-606; 610-620; 627-634; 640-664; 670-707; 714-719; 722-735; 740-741; 743-779; 808-823; 825-834; 838-850; 855-864; 868-889; 892-902; 908-909; 914-921; 924-925; 927-932; 935-942; 944-952; 961-965; 968-986; 989-993; 995-1010; 1012-1034; 1043-1063; 1074-1080; 1091-1104; 1111-1121; 1123-1128; 1134-1138; 1142-1156; 1159-1175; 1187-1190; 1192-1199; 1202-1220; 1249-1253; 1258-1262; 1264-1269; 1271-1287; 1292-1301; 1303-1309; 1315-1323; 1328-1337; 1340-1341; 1344-1361; 1365-1377; 1379-1390; 1393-1394; 1396-1398; 1419-1432; 1434-1452; 1455-1456; 1460-1465; 1468-1491; 1499; 1502; 1505-1521; 1523-1527; 1529-1532; 1536-1539; 1542-1562; 1567-1571; 1573-1582; 1587-1592; 1595-1620; 1625-1644; 1647-1654; 1659-1669; 1671-1673; 1675-1680; 1682-1686; 1688-1700; 1706-1709; 1714-1726; 1728-1734; 1738-1742; 1744-1753; 1757-1760; 1763-1764; 1766-1768; 1770-1780; 1782-1784; 1786-1789; 1791-1804; 1806-1812; 1814-1837; 1847-1856; 1858-1862; 1864-1873; 1876-1882; 1885-1896; 1902-1910; 1913-1916; 1921-1928; 1931-1936; 1940-1941; 1944-1946, or SEQ ID NO: 2N−1, wherein N=974-1101, and include sequences which are complementary to any of the above nucleotide sequences. Related nucleic acid molecules also include nucleotide sequences encoding a polypeptide comprising or consisting essentially of a substitution, modification, addition and/or deletion of one or more amino acid residues compared to the polypeptide as set forth in any of SEQ ID NO: 2N, wherein N=1-229, SEQ ID NO: 467; 488-490; 501-503; 505; 512-515; 521-522; 525-526; 528; 530; 534-537; 540; 558-559; 569; 587-594; 597; 607-609; 621-626; 635-639; 665-669; 708-713; 720-721; 736-739; 742; 780-807; 824; 835-837; 851-854; 865-867; 890-891; 903-907; 910-913; 922-923; 926; 933-934; 943; 953-960; 966-967; 987-988; 994; 1011; 1035-1042; 1064-1073; 1081-1090; 1105-1110; 1122; 1129-1133; 1139-1141; 1157-1158; 1176-1186; 1191; 1200-1201; 1221-1248; 1254-1257; 1263; 1270; 1288-1291; 1302; 1310-1314; 1324-1327; 1338-1339; 1342-1343; 1362-1364; 1378; 1391-1392; 1395; 1399-1418; 1433; 1453-1454; 1457-1459; 1466-1467; 1492-1498; 1500-1501; 1503-1504; 1522; 1528; 1533-1535; 1540-1541; 1563-1566; 1572; 1583-1586; 1593-1594; 1621-1624; 1645-1646; 1655-1658; 1670; 1674; 1681; 1687; 1701-1705; 1710-1713; 1727; 1735-1737; 1743; 1754-1756; 1761-1762; 1765; 1769; 1781; 1785; 1790; 1805; 1813; 1838-1846; 1857; 1863; 1874-1875; 1883-1884; 1897-1901; 1911-1912; 1917-1920; 1929-1930; 1937-1939; 1942-1943; or SEQ ID NO: 2N, wherein N=974-1101. Such related polypeptides may comprise, for example, additions and/or deletions of one or more N-linked or O-linked glycosylation sites, or an addition and/or a deletion of one or more cysteine residues.

For example, Table 1 illustrates, e.g., that the codons AGC, AGT, TCA, TCC, TCG, and TCT all encode the same amino acid: serine. Accordingly, at each position in the sequence where there is a codon encoding serine, any of the above trinucleotide sequences can be used without altering the encoded polypeptide.

TABLE 1
Amino acid Possible Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C TGC TGT
Aspartic acid Asp D GAC GAT
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F TTC TTT
Glycine Gly G GGA GGC GGG GGT
Histidine His H CAC CAT
Isoleucine Ile I ATA ATC ATT
Lysine Lys K AAA AAG
Leucine Leu L TTA TTG CTA CTC CTG CTT
Methionine Met M ATG
Asparagine Asn N AAC AAT
Proline Pro P CCA CCC CCG CCT
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGT
Serine Ser S AGC AGT TCA TCC TCG TCT
Threonine Thr T ACA ACC ACG ACT
Valine Val V GTA GTC GTG GTT
Tryptophan Trp W TGG
Tyrosine Tyr Y TAC TAT

Sequence alterations that do not change the amino acid sequence encoded by the polynucleotide are termed “silent” variations. With the exception of the codons ATG and TGG, encoding methionine and tryptophan, respectively, any of the possible codons for the same amino acid can be substituted by a variety of techniques, e.g., site-directed mutagenesis, available in the art. Accordingly, any and all such variations of a sequence selected from the above table are a feature of the invention.

In addition to silent variations, other conservative variations that alter one, or a few amino acids in the encoded polypeptide, can be made without altering the function of the polypeptide, these conservative variants are, likewise, a feature of the invention.

For example, substitutions, deletions and insertions introduced into the sequences provided in the Sequence Listing (except CBF polypeptide sequences SEQ ID NOs: 1956, 1958, 1960, or 2204, listed therein), are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (Wu (ed.) Methods Enzymol. (1993) vol. 217, Academic Press) or the other methods noted below. Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof can be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure. Preferably, the polypeptide encoded by the DNA performs the desired function.

Conservative substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 2 when it is desired to maintain the activity of the protein. Table 2 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as conservative substitutions.

TABLE 2
Conservative
Residue Substitutions
Ala Ser
Arg Lys
Asn Gln; His
Asp Glu
Gln Asn
Cys Ser
Glu Asp
Gly Pro
His Asn; Gln
Ile Leu; Val
Leu Ile; Val
Lys Arg; Gln
Met Leu; Ile
Phe Met; Leu; Tyr
Ser Thr; Gly
Thr Ser; Val
Trp Tyr
Tyr Trp; Phe
Val Ile; Leu

Similar substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the Table 3 when it is desired to maintain the activity of the protein. Table 3 shows amino acids which can be substituted for an amino acid in a protein and which are typically regarded as structural and functional substitutions. For example, a residue in column 1 of Table 3 may be substituted with a residue in column 2; in addition, a residue in column 2 of Table 3 may be substituted with the residue of column 1.

TABLE 3
Residue Similar Substitutions
Ala Ser; Thr; Gly; Val; Leu; Ile
Arg Lys; His; Gly
Asn Gln; His; Gly; Ser; Thr
Asp Glu: Ser; Thr
Gln Asn; Ala
Cys Ser; Gly
Glu Asp
Gly Pro; Arg
His Asn; Gln; Tyr; Phe; Lys; Arg
Ile Ala; Leu; Val; Gly; Met
yLeu Ala; Ile; Val; Gly; Met
Lys Arg; His; Gln; Gly; Pro
Met Leu; Ile; Phe
Phe Met; Leu; Tyr; Trp; His; Val; Ala
Ser Thr; Gly; Asp; Ala; Val; Ile; His
Thr Ser; Val; Ala; Gly
Trp Tyr; Phe; His
Tyr Trp; Phe; His
Val Ala; Ile; Leu; Gly; Thr; Ser; Glu

Substitutions that are less conservative than those in Table 2 can be selected by picking residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

Further Modifying Sequences of the Invention—Mutation/Forced Evolution

In addition to generating silent or conservative substitutions as noted, above, the present invention optionally includes methods of modifying the sequences of the Sequence Listing. In the methods, nucleic acid or protein modification methods are used to alter the given sequences to produce new sequences and/or to chemically or enzymatically modify given sequences to change the properties of the nucleic acids or proteins.

Thus, in one embodiment, given nucleic acid sequences are modified, e.g., according to standard mutagenesis or artificial evolution methods to produce modified sequences. The modified sequences may be created using purified natural polynucleotides isolated from any organism or may be synthesized from purified compositions and chemicals using chemical means well know to those of skill in the art. For example, Ausubel, supra, provides additional details on mutagenesis methods. Artificial forced evolution methods are described, for example, by Stemmer (1994) Nature 370: 389-391, Stemmer (1994) Proc. Natl. Acad. Sci. 91: 10747-10751, and U.S. Pat. Nos. 5,811,238, 5,837,500, and 6,242,568. Methods for engineering synthetic transcription factors and other polypeptides are described, for example, by Zhang et al. (2000) J. Biol. Chem. 275: 33850-33860, Liu et al. (2001) J. Biol. Chem. 276: 11323-11334, and Isalan et al. (2001) Nature Biotechnol. 19: 656-660. Many other mutation and evolution methods are also available and expected to be within the skill of the practitioner.

Similarly, chemical or enzymatic alteration of expressed nucleic acids and polypeptides can be performed by standard methods. For example, sequence can be modified by addition of lipids, sugars, peptides, organic or inorganic compounds, by the inclusion of modified nucleotides or amino acids, or the like. For example, protein modification techniques are illustrated in Ausubel, supra. Further details on chemical and enzymatic modifications can be found herein. These modification methods can be used to modify any given sequence, or to modify any sequence produced by the various mutation and artificial evolution modification methods noted herein.

Accordingly, the invention provides for modification of any given nucleic acid by mutation, evolution, chemical or enzymatic modification, or other available methods, as well as for the products produced by practicing such methods, e.g., using the sequences herein as a starting substrate for the various modification approaches.

For example, optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be used e.g., to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced using a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for Saccharomyces cerevisiae and mammals are TAA and TGA, respectively. The preferred stop codon for monocotyledonous plants is TGA, whereas insects and E. coli prefer to use TAA as the stop codon.

The polynucleotide sequences of the present invention can also be engineered in order to alter a coding sequence for a variety of reasons, including but not limited to, alterations which modify the sequence to facilitate cloning, processing and/or expression of the gene product. For example, alterations are optionally introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.

Furthermore, a fragment or domain derived from any of the polypeptides of the invention can be combined with domains derived from other transcription factors or synthetic domains to modify the biological activity of a transcription factor. For instance, a DNA-binding domain derived from a transcription factor of the invention can be combined with the activation domain of another transcription factor or with a synthetic activation domain. A transcription activation domain assists in initiating transcription from a DNA-binding site. Examples include the transcription activation region of VP16 or GAL4 (Moore et al. (1998) Proc. Natl. Acad. Sci. 95: 376-381; Aoyama et al. (1995) Plant Cell 7: 1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51: 113-119) and synthetic peptides (Giniger and Ptashne (1987) Nature 330: 670-672).

Expression and Modification of Polypeptides

Typically, polynucleotide sequences of the invention are incorporated into recombinant DNA (or RNA) molecules that direct expression of polypeptides of the invention in appropriate host cells, transgenic plants, in vitro translation systems, or the like. Due to the inherent degeneracy of the genetic code, nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can be substituted for any listed sequence to provide for cloning and expressing the relevant homolog.

The transgenic plants of the present invention comprising recombinant polynucleotide sequences are generally derived from parental plants, which may themselves be non-transformed (or non-transgenic) plants. These transgenic plants may either have a transcription factor gene “knocked out” (for example, with a genomic insertion by homologous recombination, an antisense or ribozyme construct) or expressed to a normal or wild-type extent. However, overexpressing transgenic “progeny” plants will exhibit greater mRNA levels, wherein the mRNA encodes a transcription factor, that is, a DNA-binding protein that is capable of binding to a DNA regulatory sequence and inducing transcription, and preferably, expression of a plant trait gene. Preferably, the mRNA expression level will be at least three-fold greater than that of the parental plant, or more preferably at least ten-fold greater mRNA levels compared to said parental plant, and most preferably at least fifty-fold greater compared to said parental plant.

Vectors, Promoters, and Expression Systems

The present invention includes recombinant constructs comprising one or more of the nucleic acid sequences herein. The constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus (e.g., a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

General texts that describe molecular biological techniques useful herein, including the use and production of vectors, promoters and many other relevant topics, include Berger, Sambrook, supra and Ausubel, supra. Any of the identified sequences can be incorporated into a cassette or vector, e.g., for expression in plants. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, Academic Press, and Gelvin et al. (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed by Herrera-Estrella et al. (1983) Nature 303: 209, Bevan (1984) Nucleic Acids Res. 12: 8711-8721, Klee (1985) Bio/Technology 3: 637-642, for dicotyledonous plants.

Alternatively, non-Ti vectors can be used to transfer the DNA into monocotyledonous plants and cells by using free DNA delivery techniques. Such methods can involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou (1991) Bio/Technology 9: 957-962) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be produced. An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993) Plant Physiol. 102: 1077-1084; Vasil (1993) Bio/Technology 10: 667-674; Wan and Lemeaux (1994) Plant Physiol. 104: 37-48, and for Agrobacterium-mediated DNA transfer (Ishida et al. (1996) Nature Biotechnol. 14: 745-750).

Typically, plant transformation vectors include one or more cloned plant coding sequence (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.

A potential utility for the transcription factor polynucleotides disclosed herein is the isolation of promoter elements from these genes that can be used to program expression in plants of any genes. Each transcription factor gene disclosed herein is expressed in a unique fashion, as determined by promoter elements located upstream of the start of translation, and additionally within an intron of the transcription factor gene or downstream of the termination codon of the gene. As is well known in the art, for a significant portion of genes, the promoter sequences are located entirely in the region directly upstream of the start of translation. In such cases, typically the promoter sequences are located within 2.0 kb of the start of translation, or within 1.5 kb of the start of translation, frequently within 1.0 kb of the start of translation, and sometimes within 0.5 kb of the start of translation.

The promoter sequences can be isolated according to methods known to one skilled in the art.

Examples of constitutive plant promoters which can be useful for expressing the TF sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odell et al. (1985) Nature 313: 810-812); the nopaline synthase promoter (An et al. (1988) Plant Physiol. 88: 547-552); and the octopine synthase promoter (Fromm et al. (1989) Plant Cell 1: 977-984).

A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a TF sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like. Numerous known promoters have been characterized and can favorably be employed to promote expression of a polynucleotide of the invention in a transgenic plant or cell of interest. For example, tissue specific promoters include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (Bird et al. (1988) Plant Mol. Biol. 11: 651-662), root-specific promoters, such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186, pollen-active promoters such as PTA29, PTA26 and PTA13 (U.S. Pat. No. 5,792,929), promoters active in vascular tissue (Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988), flower-specific (Kaiser et al. (1995) Plant Mol. Biol. 28: 231-243), pollen (Baerson et al. (1994) Plant Mol. Biol. 26: 1947-1959), carpels (Ohl et al. (1990) Plant Cell 2: 837-848), pollen and ovules (Baerson et al. (1993) Plant Mol. Biol. 22: 255-267), auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol Biol. 39: 979-990 or Baumann et al. (1999) Plant Cell 11: 323-334), cytokinin-inducible promoter (Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promoters responsive to gibberellin (Shi et al. (1998) Plant Mol. Biol. 38: 1053-1060, Willmott et al. (1998) 38: 817-825) and the like. Additional promoters are those that elicit expression in response to heat (Ainley et al. (1993) Plant Mol. Biol. 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989) Plant Cell 1: 471-478, and the maize rbcS promoter, Schaffner and Sheen (1991) Plant Cell 3: 997-1012); wounding (e.g., wunI, Siebertz et al. (1989) Plant Cell 1: 961-968); pathogens (such as the PR-1 promoter described in Buchel et al. (1999) Plant Mol. Biol. 40: 387-396, and the PDF1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38: 1071-1080), and chemicals such as methyl jasmonate or salicylic acid (Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 89-108). In addition, the timing of the expression can be controlled by using promoters such as those acting at senescence (Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (Odell et al. (1994) Plant Physiol. 106: 447-458).

Plant expression vectors can also include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors can include additional regulatory sequences from the 3′-untranslated region of plant genes, e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.

Additional Expression Elements

Specific initiation signals can aid in efficient translation of coding sequences. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where a coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon can be separately provided. The initiation codon is provided in the correct reading frame to facilitate transcription. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use.

Expression Hosts

The present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention (including fragments thereof) by recombinant techniques. Host cells are genetically engineered (i.e., nucleic acids are introduced, e.g., transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector comprising the relevant nucleic acids herein. The vector is optionally a plasmid, a viral particle, a phage, a naked nucleic acid, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the relevant gene. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, Sambrook, supra and Ausubel, supra.

The host cell can be a eukaryotic cell, such as a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Plant protoplasts are also suitable for some applications. For example, the DNA fragments are introduced into plant tissues, cultured plant cells or plant protoplasts by standard methods including electroporation (Fromm et al. (1985) Proc. Natl. Acad. Sci. 82: 5824-5828, infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors Academic Press, New York, N.Y., pp. 549-560; U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al. (1987) Nature 327: 70-73), use of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al. (1984) Science 233: 496-498; Fraley et al. (1983) Proc. Natl. Acad. Sci. 80: 4803-4807).

The cell can include a nucleic acid of the invention that encodes a polypeptide, wherein the cell expresses a polypeptide of the invention. The cell can also include vector sequences, or the like. Furthermore, cells and transgenic plants that include any polypeptide or nucleic acid above or throughout this specification, e.g., produced by transduction of a vector of the invention, are an additional feature of the invention.

For long-term, high-yield production of recombinant proteins, stable expression can be used. Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides encoding mature proteins of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.

Modified Amino Acid Residues

Polypeptides of the invention may contain one or more modified amino acid residues. The presence of modified amino acids may be advantageous in, for example, increasing polypeptide half-life, reducing polypeptide antigenicity or toxicity, increasing polypeptide storage stability, or the like. Amino acid residue(s) are modified, for example, co-translationally or post-translationally during recombinant production or modified by synthetic or chemical means.

Non-limiting examples of a modified amino acid residue include incorporation or other use of acetylated amino acids, glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, PEG modified (e.g., “PEGylated”) amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, etc. References adequate to guide one of skill in the modification of amino acid residues are replete throughout the literature.

The modified amino acid residues may prevent or increase affinity of the polypeptide for another molecule, including, but not limited to, polynucleotide, proteins, carbohydrates, lipids and lipid derivatives, and other organic or synthetic compounds.

Identification of Additional Factors

A transcription factor provided by the present invention can also be used to identify additional endogenous or exogenous molecules that can affect a phentoype or trait of interest. On the one hand, such molecules include organic (small or large molecules) and/or inorganic compounds that affect expression of (i.e., regulate) a particular transcription factor. Alternatively, such molecules include endogenous molecules that are acted upon either at a transcriptional level by a transcription factor of the invention to modify a phenotype as desired. For example, the transcription factors can be employed to identify one or more downstream genes that are subject to a regulatory effect of the transcription factor. In one approach, a transcription factor or transcription factor homolog of the invention is expressed in a host cell, e.g., a transgenic plant cell, tissue or explant, and expression products, either RNA or protein, of likely or random targets are monitored, e.g., by hybridization to a microarray of nucleic acid probes corresponding to genes expressed in a tissue or cell type of interest, by two-dimensional gel electrophoresis of protein products, or by any other method known in the art for assessing expression of gene products at the level of RNA or protein. Alternatively, a transcription factor of the invention can be used to identify promoter sequences (such as binding sites on DNA sequences) involved in the regulation of a downstream target. After identifying a promoter sequence, interactions between the transcription factor and the promoter sequence can be modified by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter a plant trait. Typically, transcription factor DNA-binding sites are identified by gel shift assays. After identifying the promoter regions, the promoter region sequences can be employed in double-stranded DNA arrays to identify molecules that affect the interactions of the transcription factors with their promoters (Bulyk et al. (1999) Nature Biotechnol. 17: 573-577).

The identified transcription factors are also useful to identify proteins that modify the activity of the transcription factor. Such modification can occur by covalent modification, such as by phosphorylation, or by protein-protein (homo or -heteropolymer) interactions. Any method suitable for detecting protein-protein interactions can be employed. Among the methods that can be employed are co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns, and the two-hybrid yeast system.

The two-hybrid system detects protein interactions in vivo and is described in Chien et al. (1991) Proc. Natl. Acad. Sci. 88: 9578-9582, and is commercially available from Clontech (Palo Alto, Calif.). In such a system, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the TF polypeptide and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into the plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. Then, the library plasmids responsible for reporter gene expression are isolated and sequenced to identify the proteins encoded by the library plasmids. After identifying proteins that interact with the transcription factors, assays for compounds that interfere with the TF protein-protein interactions can be preformed.

Identification of Modulators

In addition to the intracellular molecules described above, extracellular molecules that alter activity or expression of a transcription factor, either directly or indirectly, can be identified. For example, the methods can entail first placing a candidate molecule in contact with a plant or plant cell. The molecule can be introduced by topical administration, such as spraying or soaking of a plant, or incubating a plant in a solution containing the molecule, and then the molecule's effect on the expression or activity of the TF polypeptide or the expression of the polynucleotide monitored. Changes in the expression of the TF polypeptide can be monitored by use of polyclonal or monoclonal antibodies, gel electrophoresis or the like. Changes in the expression of the corresponding polynucleotide sequence can be detected by use of microarrays, Northerns, quantitative PCR, or any other technique for monitoring changes in mRNA expression. These techniques are exemplified in Ausubel et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons (1998, and supplements through 2001).Changes in the activity of the transcription factor can be monitored, directly or indirectly, by assaying the function of the transcription factor, for example, by measuring the expression of promoters known to be controlled by the transcription factor (using promoter-reporter constructs), measuring the levels of transcripts using microarrays, Northern blots, quantitative PCR, etc. Such changes in the expression levels can be correlated with modified plant traits and thus identified molecules can be useful for soaking or spraying on fruit, vegetable and grain crops to modify traits in plants.

Essentially any available composition can be tested for modulatory activity of expression or activity of any nucleic acid or polypeptide herein. Thus, available libraries of compounds such as chemicals, polypeptides, nucleic acids and the like can be tested for modulatory activity. Often, potential modulator compounds can be dissolved in aqueous or organic (e.g., DMSO-based) solutions for easy delivery to the cell or plant of interest in which the activity of the modulator is to be tested. Optionally, the assays are designed to screen large modulator composition libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microplates in robotic assays).

In one embodiment, high throughput screening methods involve providing a combinatorial library containing a large number of potential compounds (potential modulator compounds). Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as target compounds.

A combinatorial chemical library can be, e.g., a collection of diverse chemical compounds generated by chemical synthesis or biological synthesis. For example, a combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (e.g., in one example, amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound of a set length). Exemplary libraries include peptide libraries, nucleic acid libraries, antibody libraries (see, e.g., Vaughn et al. (1996) Nature Biotechnol. 14: 309-314 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. Science (1996) 274: 1520-1522 and U.S. Pat. No. 5,593,853), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), and small organic molecule libraries (see, e.g., benzodiazepines, in Baum Chem. & Engineering News Jan. 18, 1993, page 33; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337) and the like.

Preparation and screening of combinatorial or other libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, (1991) Int. J. Pept. Prot. Res. 37: 487-493; and Houghton et al. (1991) Nature 354: 84-88). Other chemistries for generating chemical diversity libraries can also be used.

In addition, as noted, compound screening equipment for high-throughput screening is generally available, e.g., using any of a number of well known robotic systems that have also been developed for solution phase chemistries useful in assay systems. These systems include automated workstations including an automated synthesis apparatus and robotic systems utilizing robotic arms. Any of the above devices are suitable for use with the present invention, e.g., for high-throughput screening of potential modulators. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.

Indeed, entire high-throughput screening systems are commercially available. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. Similarly, microfluidic implementations of screening are also commercially available.

The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. The integrated systems herein, in addition to providing for sequence alignment and, optionally, synthesis of relevant nucleic acids, can include such screening apparatus to identify modulators that have an effect on one or more polynucleotides or polypeptides according to the present invention.

In some assays it is desirable to have positive controls to ensure that the components of the assays are working properly. At least two types of positive controls are appropriate. That is, known transcriptional activators or inhibitors can be incubated with cells or plants, for example, in one sample of the assay, and the resulting increase/decrease in transcription can be detected by measuring the resulting increase in RNA levels and/or protein expression, for example, according to the methods herein. It will be appreciated that modulators can also be combined with transcriptional activators or inhibitors to find modulators that inhibit transcriptional activation or transcriptional repression. Either expression of the nucleic acids and proteins herein or any additional nucleic acids or proteins activated by the nucleic acids or proteins herein, or both, can be monitored.

In an embodiment, the invention provides a method for identifying compositions that modulate the activity or expression of a polynucleotide or polypeptide of the invention. For example, a test compound, whether a small or large molecule, is placed in contact with a cell, plant (or plant tissue or explant), or composition comprising the polynucleotide or polypeptide of interest and a resulting effect on the cell, plant, (or tissue or explant) or composition is evaluated by monitoring, either directly or indirectly, one or more of: expression level of the polynucleotide or polypeptide, activity (or modulation of the activity) of the polynucleotide or polypeptide. In some cases, an alteration in a plant phenotype can be detected following contact of a plant (or plant cell, or tissue or explant) with the putative modulator, e.g., by modulation of expression or activity of a polynucleotide or polypeptide of the invention. Modulation of expression or activity of a polynucleotide or polypeptide of the invention may also be caused by molecular elements in a signal transduction second messenger pathway and such modulation can affect similar elements in the same or another signal transduction second messenger pathway.

Subsequences

Also contemplated are uses of polynucleotides, also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 15, more preferably at least 20, 30, or 50 bases, which hybridize under at least highly stringent (or ultra-high stringent or ultra-ultra-high stringent conditions) conditions to a polynucleotide sequence described above. The polynucleotides may be used as probes, primers, sense and antisense agents, and the like, according to methods as noted supra.

Subsequences of the polynucleotides of the invention, including polynucleotide fragments and oligonucleotides are useful as nucleic acid probes and primers. An oligonucleotide suitable for use as a probe or primer is at least about 15 nucleotides in length, more often at least about 18 nucleotides, often at least about 21 nucleotides, frequently at least about 30 nucleotides, or about 40 nucleotides, or more in length. A nucleic acid probe is useful in hybridization protocols, e.g., to identify additional polypeptide homologs of the invention, including protocols for microarray experiments. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods. See Sambrook, supra, and Ausubel, supra.

In addition, the invention includes an isolated or recombinant polypeptide including a subsequence of at least about 15 contiguous amino acids encoded by the recombinant or isolated polynucleotides of the invention. For example, such polypeptides, or domains or fragments thereof, can be used as immunogens, e.g., to produce antibodies specific for the polypeptide sequence, or as probes for detecting a sequence of interest. A subsequence can range in size from about 15 amino acids in length up to and including the full length of the polypeptide.

To be encompassed by the present invention, an expressed polypeptide which comprises such a polypeptide subsequence performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. For example, a polypeptide fragment can comprise a recognizable structural motif or functional domain such as a DNA binding domain that activates transcription, e.g., by binding to a specific DNA promoter region an activation domain, or a domain for protein-protein interactions.

Production of Transgenic Plants

Modification of Traits

The polynucleotides of the invention are favorably employed to produce transgenic plants with various traits, or characteristics, that have been modified in a desirable manner, e.g., to improve the seed characteristics of a plant. For example, alteration of expression levels or patterns (e.g., spatial or temporal expression patterns) of one or more of the transcription factors (or transcription factor homologs) of the invention, as compared with the levels of the same protein found in a wild-type plant, can be used to modify a plant's traits. An illustrative example of trait modification, improved characteristics, by altering expression levels of a particular transcription factor is described further in the Examples and the Sequence Listing.

Arabidopsis as a Model System

Arabidopsis thaliana is the object of rapidly growing attention as a model for genetics and metabolism in plants. Arabidopsis has a small genome, and well-documented studies are available. It is easy to grow in large numbers and mutants defining important genetically controlled mechanisms are either available, or can readily be obtained. Various methods to introduce and express isolated homologous genes are available (see Koncz et al. eds., et al. Methods in Arabidopsis Research (1992) et al. World Scientific, New Jersey, NJ, in “Preface”). Because of its small size, short life cycle, obligate autogamy and high fertility, Arabidopsis is also a choice organism for the isolation of mutants and studies in morphogenetic and development pathways, and control of these pathways by transcription factors (Koncz supra, p. 72). A number of studies introducing transcription factors into A. thaliana have demonstrated the utility of this plant for understanding the mechanisms of gene regulation and trait alteration in plants. (See, for example, Koncz supra, and U.S. Pat. No. 6,417,428).

Arabidopsis Genes in Transgenic Plants.

Expression of genes which encode transcription factors modify expression of endogenous genes, polynucleotides, and proteins are well known in the art. In addition, transgenic plants comprising isolated polynucleotides encoding transcription factors may also modify expression of endogenous genes, polynucleotides, and proteins. Examples include Peng et al. (1997) et al. Genes and Development 11: 3194-3205, and Peng et al. (1999) Nature 400: 256-261. In addition, many others have demonstrated that an Arabidopsis transcription factor expressed in an exogenous plant species elicits the same or very similar phenotypic response. See, for example, Fu et al. (2001) Plant Cell 13: 1791-1802; Nandi et al. (2000) Curr. Biol. 10: 215-218; Coupland (1995) Nature 377: 482-483; and Weigel and Nilsson (1995) Nature 377: 482-500.

Homologous Genes Introduced into Transgenic Plants.

Homologous genes that may be derived from any plant, or from any source whether natural, synthetic, semi-synthetic or recombinant, and that share significant sequence identity or similarity to those provided by the present invention, may be introduced into plants, for example, crop plants, to confer desirable or improved traits. Consequently, transgenic plants may be produced that comprise a recombinant expression vector or cassette with a promoter operably linked to one or more sequences homologous to presently disclosed sequences. The promoter may be, for example, a plant or viral promoter.

The invention thus provides for methods for preparing transgenic plants, and for modifying plant traits. These methods include introducing into a plant a recombinant expression vector or cassette comprising a functional promoter operably linked to one or more sequences homologous to presently disclosed sequences. Plants and kits for producing these plants that result from the application of these methods are also encompassed by the present invention.

Transcription Factors of Interest for the Modification of Plant Traits

Currently, the existence of a series of maturity groups for different latitudes represents a major barrier to the introduction of new valuable traits. Any trait (e.g. disease resistance) has to be bred into each of the different maturity groups separately, a laborious and costly exercise. The availability of single strain, which could be grown at any latitude, would therefore greatly increase the potential for introducing new traits to crop species such as soybean and cotton.

For many of the specific effects, traits and utilities listed in Table 4 and Table 6 that may be conferred to plants, one or more transcription factor genes may be used to increase or decrease, advance or delay, or improve or prove deleterious to a given trait. Overexpressing or suppressing one or more genes can impart significant differences in production of plant products, such as different fatty acid ratios. For example, overexpression of G720 caused a plant to become more freezing tolerant, but knocking out the same transcription factor imparted greater susceptibility to freezing. Thus, suppressing a gene that causes a plant to be more sensitive to cold may improve a plant's tolerance of cold. More than one transcription factor gene may be introduced into a plant, either by transforming the plant with one or more vectors comprising two or more transcription factors, or by selective breeding of plants to yield hybrid crosses that comprise more than one introduced transcription factor.

A listing of specific effects and utilities that the presently disclosed transcription factor genes have on plants, as determined by direct observation and assay analysis, is provided in Table 4. Table 4 shows the polynucleotides identified by SEQ ID NO; Mendel Gene ID No. (GID); and if the polynucleotide was tested in a transgenic assay. The first column shows the polynucleotide SEQ ID NO; the second column shows the GID; the third column shows whether the gene was overexpressed (OF) or knocked out (KO) in plant studies; the fourth column shows the trait(s) resulting from the knock out or overexpression of the polynucleotide in the transgenic plant; the fifth column shows the category of the trait; and the sixth column (“Comment”), includes specific observations made with respect to the polynucleotide of the first column.

TABLE 4
Traits, trait categories, and effects and utilities that
transcription factor genes have on plants.
Polynucleotide GID OE/
SEQ ID NO: No. KO Trait(s) Category Observations
1 G8 OE Flowering time Flowering time Late flowering
3 G19 OE Erysiphe Disease Increased tolerance to Erysiphe;
repressed by methyl jasmonate and
induced by 1-aminocyclopropane 1-
carboxylic acid (ACC)
5 G22 OE Sodium chloride Abiotic stress Increased tolerance to high salt
7 G24 OE Morphology: other Dev and morph Reduced size and necrotic patches
9 G28 OE Botrytis Disease Increased tolerance to Botrytis
Selerotinia Disease Increased tolerance to Sclerotinia
Erysiphe Disease Increased resistance to Erysiphe
11 G47 OE Stem Dev and morph Altered structure of vascular tissues
Osmotic Abiotic stress Better root growth under osmotic
Flowering time Flowering time stress
Architecture Dev and morph Late flowering
Altered architecture and
Architecture Dev and morph inflorescence development
Reduced apical dominance
13 G156 KO Seed Dev and morph Seed color alteration
15 G157 OB Flowering time Flowering time Altered flowering time (modest level
of overexpression triggers early
flowering, whereas a larger increase
delays flowering)
17 G162 OE Seed oil content Seed biochemistry Increased seed oil content
Seed protein content Seed biochemistry Increased seed protein content
19 G175 OE Osmotic Abiotic stress Increased tolerance to osmotic stress
21 G180 OE Seed oil content Seed biochemistry Decreased seed oil
Flowering time Flowering time Early flowering
23 G183 OE Flowering time Flowering time Early flowering
Light response Dev and morph Constitutive photomorphogenesis
25 G188 KO Fusarium Disease Increased susceptibility to Fusarium
Osmotic Abiotic stress Better germination under osmotic
stress
27 G189 OE Size Dev and morph Increased leaf size
29 G192 OE Flowering time Flowering time Late flowering
Seed oil content Seed biochemistry Decreased seed oil content
31 G196 OE Sodium chloride Abiotic stress Increased tolerance to high salt
33 G211 OE Leaf insoluble sugars Leaf biochemistry Increase in leaf xylose
Architecture Dev and morph Reduced apical dominance
Leaf Dev and morph Altered leaf shape
35 G214 OB Flowering time Flowering time Late flowering
Leaf fatty acids Leaf biochemistry Increased leaf fatty acids
Seed prenyl lipids Seed biochemistry Increased seed lutein
Leaf prenyl lipids Leaf biochemistry Increased leaf chlorophyll and
carotenoids
37 G226 OE Seed protein content Seed biochemistry Increased seed protein
Trichome Dev and morph Glabrous, lack of trichomes
Root Dev and morph Increased root hairs
Sodium chloride Abiotic stress Increased tolerance to high salt
Nutrient uptake Abiotic stress Increased tolerance to nitrogen-
limited medium
39 G241 KO Seed protein content Seed biochemistry Increased seed protein content
Seed oil content Seed biochemistry Decreased seed oil
Sugar sensing Sugar sensing Decreased germination and growth
on glucose medium
41 G248 OE Botrytis Disease Increased susceptibility to Botrytis
43 G254 OE Sugar sensing Sugar sensing Decreased germination and growth
45 G256 OE Cold, chilling Abiotic stress Better germination and growth in cold
47 G278 OE Sclerotinia Disease Increased susceptibility to
Sclerotinia
49 G291 OE Seed oil content Seed biochemistry Increased seed oil content
51 G303 OE Osmotic Abiotic stress Better germination on high sucrose
and high NaCl
53 G312 OE Sodium chloride Abiotic stress Better germination on high NaCl
55 G325 OE Osmotic Abiotic stress Better germination on high sucrose
and NaCl
57 G343 OE Glyphosate Herbicide sensitivity Increased resistance to glyphosate
Size Dev and morph Small plant
59 G353 OE Osmotic Abiotic stress Increased seedling vigor on
polyethylene glycol (PEG)
Size Dev and morph Reduced size
Leaf Dev and morph Altered leaf development
Flower Dev and morph Short pedicels, downward pointing
siligues
61 G354 OE Size Dev and morph Reduced size
Light response Dev and morph Constitutive photomorphogenesis
Flower Dev and morph Short pedicels, downward pointing
siligues
63 G361 OE Flowering time Flowering time Late flowering
65 G362 OE Flowering time Flowering time Late flowering
Size Dev and morph Reduced size
Trichome Dev and morph Ectopic trichome formation,
increased trichome number
Morphology: other Dev and morph Increased pigmentation in seed and
embryos, and in other organs
67 G371 OE Botrytis Disease Increased susceptibility to Botrytis
69 G390 OE Architecture Dev and morph Altered shoot development
71 G391 OE Architecture Dev and morph Altered shoot development
73 G409 OE Erysiphe Disease Increased tolerance to Erysiphe
75 G427 OE Seed oil content Seed biochemistry Increased oil content
Seed protein content Seed biochemistry Decreased protein content
77 G438 KO Stem Dev and morph Reduced lignin
Architecture Dev and morph Reduced branching
79 G450 OE Seed Dev and morph Increased seed size
81 G464 OE Heat Abiotic stress Better germination and growth in heat
83 G470 OE Fertility Dev and morph Short stamen filaments
85 G477 OE Sclerotinia Disease Increased susceptibility to
Oxidative Abiotic stress Sclerotinia
Increased sensitivity to oxidative
stress
87 G481 OE Sugar sensing Sugar sensing Better germination on sucrose media
Drought Abiotic stress Increased tolerance to drought
89 G482 OE Sodium chloride Abiotic stress Increased tolerance to high salt
91 G484 KO Seed glucosinolates Seed biochemistry Altered glucosinolate profile
93 G489 OE Osmotic Abiotic stress Increased tolerance to osmotic stress
95 G490 OE Flowering time Flowering time Early flowering
97 G504 OE Seed oil composition Seed biochemistry Decreased seed oil composition and
content; increase in 18:2 fatty acid
and decrease in 20:1 fat acid
99 G509 KO Seed oil content Seed biochemistry Increased total seed oil and protein
Seed protein content Seed biochemistry content
101 G519 OE Seed oil content Seed biochemistry Increased seed oil content
103 G545 OE Sodium chloride Abiotic stress Susceptible to high salt
Erysiphe Disease Increased susceptibility to Erysiphe
Pseudomonas Disease Increased susceptibility to Pseudomonas
Fusarium Disease Increased susceptibility to Fusarium
Nutrient uptake Abiotic stress Increased tolerance to phosphate-free
medium
105 G546 OE Hormone sensitivity Hormone sensitivity Decreased sensitivity to abscisic acid (ABA)
107 G561 OE Seed oil content Seed biochemistry Increased seed oil content
Nutrient uptake Abiotic stress Increased tolerance to potassium-free
medium
109 G562 OE Flowering time Flowering time Late flowering
111 G567 OE Seed oil content Seed biochemistry Increased total seed oil/protein
Seed protein content Seed biochemistry content
Sugar sensing Sugar sensing Increased total seed oil/protein
content
Decreased seedling vigor on high
glucose
113 G568 OE Architecture Dev and morph Altered branching
115 G584 OE Seed Dev and morph Large seeds
117 G585 OE Trichome Dev and morph Reduced trichome density
119 G590 KO Seed oil content Seed biochemistry Increased seed oil content
OE Flowering time Flowering time Early flowering
121 G594 OE Sclerotinia Disease Increased susceptibility to
Sclerotinia
123 G597 OE Seed protein content Seed biochemistry Altered seed protein content
125 G598 OE Seed oil content Seed biochemistry Increased seed oil
127 G634 OE Trichome Dev and morph Increased trichome density and size
129 G635 OE Variegation Dev and morph Altered coloration
131 G636 OE Senescence Dev and morph Premature senescence
133 G638 OE Flower Dev and morph Altered flower development
135 G652 KO Seed prenyl lipids Seed biochemistry Increase in alpha-tocopherol
137 G663 OE Biochemistry: other Biochem: misc Increased anthocyanins in leaf, root, seed
139 G664 OE Cold, chilling Abiotic stress Better germination and growth in cold
141 G674 OE Leaf Dev and morph Dark green, upwardly oriented leaves
143 G676 OE Trichome Dev and morph Reduced trichome number, ectopic
trichome formation
145 G680 OE Sugar sensing Sugar sensing Reduced germination on glucose medium
147 G682 OE Trichome Dev and morph Glabrous, lack of trichomes
Heat Abiotic stress Better germination and growth in
Root Dev and morph heat
Increased root hairs
149 G715 OE Seed oil content Seed biochemistry Increased seed oil content
151 G720 OE Freezing Abiotic stress More freezing tolerant
KO Freezing Abiotic stress Increased susceptibility to freezing
153 G736 OE Flowering time Flowering time Late flowering
Leaf Dev and morph Altered leaf shape
155 G748 OE Seed prenyl lipids Seed biochemistry Increased lutein content
Stem Dev and morph More vascular bundles in stem
Flowering time Flowering time Late flowering
157 G779 OE Fertility Dev and morph Reduced fertility
Flower Dev and morph Homeotic transformations
159 G789 OE Flowering time Flowering time Early flowering
161 G801 OE Sodium chloride Abiotic stress Better germination on high NaCl
163 G849 KO Seed oil content Seed biochemistry Increased seed oil content
Seed protein content Seed biochemistry Altered seed protein content
165 G859 OE Flowering time Flowering time Late flowering
167 G864 OE Heat Abiotic stress Better germination in heat
169 G867 OE Sodium chloride Abiotic stress Better seedling vigor on high salt
Sugar sensing Sugar sensing Better seedling vigor on high sucrose
171 G869 OE Seed oil composition Seed biochemistry Altered seed fatty acids
173 G877 KO Embryo lethal Dev and morph Embryo lethal phenotype: potential
herbicide target
175 G881 OE Erysiphe Disease Increased susceptibility to Erysiphe
177 G892 KO Seed protein content Seed biochemistry Altered seed protein content
Seed oil content Seed biochemistry Altered seed oil content
179 G896 KO Fusarium Disease Increased susceptibility to Fusarium
181 G910 OE Flowering time Flowering time Late flowering
183 G911 OE Nutrient uptake Abiotic stress Increased growth on potassium-free medium
185 G912 OE Freezing Abiotic stress Freezing tolerant
Drought Abiotic stress Increased survival in drought
Morphology: other Dev and morph conditions
Sugar sensing Sugar sensing Dark green color
Reduced cotyledon expansion in
glucose
187 G913 OE Freezing Abiotic stress Increased tolerance to freezing
Flowering time Flowering time Late flowering
Drought Abiotic stress Increased tolerance to drought
189 G922 OE Osmotic Abiotic stress Better germination on high sucrose
Sodium chloride Abiotic stress Better germination, increased root
growth on high salt
191 G926 KO Hormone sensitivity Hormone sensitivity Reduced sensitivity to ABA
Osmotic Abiotic stress Increased tolerance to osmotic stress
(salt and sucrose)
193 G961 KO Seed oil content Seed biochemistry Increased seed oil content
195 G971 OE Flowering time Flowering time Late flowering
197 G974 OE Seed oil content Seed biochemistry Altered seed oil content
199 G975 OE Leaf fatty acids Leaf biochemistry Increased wax in leaves
201 G979 KO Seed Dev and morph Altered seed development, ripening,
and germination
203 G987 KO Leaf fatty acids Leaf biochemistry Reduction in 16:3 fatty acids
Leaf prenyl lipids Leaf biochemistry Altered chlorophyll, tocopherol,
carotenoid
205 G988 OE Seed protein content Seed biochemistry Increased seed protein content
Flower Dev and morph Enlarged floral organs, short pedicels
Architecture Dev and morph Reduced lateral branching
Stem Dev and morph Thicker stem, altered distribution of
vascular bundles
207 G1040 OE Seed Dev and morph Smaller and more rounded seeds
209 G1047 OE Fusarium Disease Increased tolerance to Fusarium
211 G1051 OE Flowering time Flowering time Late flowering
213 G1052 OE Flowering time Flowering time Late flowering
215 G1062 KO Seed Dev and morph Altered seed shape
217 G1063 OE Leaf Dev and morph Altered leaf shape, dark green color
Inflorescence Dev and morph Altered inflorescence development
Flower Dev and morph Altered flower development, ectopic
carpel tissue
219 G1064 OE Botrytis Disease Increased sensitivity to Botrytis
221 G1069 OE Hormone sensitivity Hormone sensitivity Reduced ABA sensitivity
Osmotic Abiotic stress Better germination under osmotic
stress
223 G1073 OE Size Dev and morp Substantially increased plant size
Seed Dev and morph Increased seed yield
Drought Abiotic stress Increased tolerance to drought
225 G1075 OE Flower Dev and morph Reduced or absent petals, sepals and
stamens
227 G1064 OE Botrytis Disease Increased susceptibility to Botrytis
229 G1089 KO Osmotic Abiotic stress Better germination under osmotic
stress
231 G1134 OE Hormone sensitivity Hormone sensitivity Altered response to ethylene longer
hypocotyls and lack of apical hook
233 G1140 OE Flower Dev and morph Altered flower development
235 G1143 OE Seed oil content Seed biochemistry Altered seed oil content
237 G1146 OE Leaf Dev and morph Altered leaf development
239 G1196 KO Botrytis Disease Increased susceptibility to Botrytis
241 G1198 OE Seed oil content Seed biochemistry Increased seed oil content
243 G1225 OE Flowering time Flowering time Early flowering
Sugar sensing Sugar sensing Better germination on sucrose and
glucose media
245 G1226 OE Seed oil content Seed biochemistry Increased seed oil content
247 G1229 OE Seed oil content Seed biochemistry Decreased seed oil content
249 G1255 OE Botrytis Disease Increased susceptibility to Botrytis
Seed Dev and morph Increased seed size
Morphology: other Dev and morph Reduced apical dominance
251 G1266 OE Erysiphe Disease Increased tolerance to Erysiphe
253 G1275 OE Architecture Dev and morph Reduced apical dominance
255 G1305 OE Heat Abiotic stress Reduced chlorosis in heat
257 G1322 OE Chilling Abiotic stress Increased seedling vigor in cold
Size Dev and morph Reduced size
Leaf glucosinolates Leaf biochemistry Increase in M39480
259 G1323 OE Seed oil content Seed biochemistry Decreased seed oil
Seed protein content Seed biochemistry Increased seed protein
261 G1330 OE Hormone sensitivity Hormone sensitivity Ethylene insensitive when
germination in the dark on ACC
263 G1331 OE Light response Dev and morph Constitutive photomorphogenesis
265 G1332 OE Trichome Dev and morph Reduced trichome denisty
267 G1363 OE Fusarium Disease Increased tolerance to Fusarium
269 G1411 OE Architecture Dev and morph Loss of apical dominance
271 G1417 KO Seed oil composition Seed biochemistry Incresase in 1 & 2, decrease in 1 & 3
fatty acids
273 G1419 OE Seed protein content Seed biochemistry Increased seed protein
275 G1449 OE Flower Dev and morph Altered flower structure
277 G1451 OE Morphology: other Dev and morph Increased plant size
OE Leaf Dev and morph Large leaf size
KO Seed oil content Seed biochemistry Altered seed oil content
279 G1452 OE Trichome Dev and morph Reduced trichome density
Leaf Dev and morph Altered leaf shape, dark green color
Hormone sensitivity Hormone sensitivity Reduced sensitivity to ABA
Osmotic Abiotic stress Better germination on sucrose and
Flowering time Flowering time salt
Late flowering
281 G1463 OE Senescence Dev and morph Premature senescence
283 G1471 OE Seed oil content Seed biochemistry Increased seed oil content
285 G1478 OE Seed protein content Seed biochemistry Decreased seed protein content
Flowering time Flowering time Late flowering
Seed oil content Seed biochemistry Increased seed oil content
287 G1482 KO Biochemistry: other Biochem: misc Increased anthocyanins
OE Root Dev and morph Increased root growth
289 G1488 OE Seed protein content Seed biochemistry Altered seed protein content
Light response Dev and morph Constitutive photomorphogenesis
Architecture Dev and morph Reduced apical dominance, shorter
stems
291 G1494 OE Flowering time Flowering time Early flowering
Light response Dev and morph Long hypocotyls, altered leaf shape
Leaf Dev and morph Pale green leaves, altered leaf shape
293 G1496 OE Seed oil content Seed biochemistry Altered seed oil content
295 G1499 OE Morphology: other Dev and morph Dark green color
Architecture Dev and morph Altered plant architecture
Flower Dev and morph Altered floral organ identity and
development
297 G1519 KO Embryo lethal Dev and morph Embryo lethal phenotype: potential
herbicide target
299 G1526 KO Seed oil content Seed biochemistry Increased seed oil content
301 G1540 OE Morphology: other Dev and morph Reduced cell differentiation in
303 G1543 OE Architectur e Dev and morph Altered architecture, compact plant
Morphology: other Dev and morph Dark green color
Seed oil content Seed biochemistry Decreased seed oil
Leaf prenyl lipids Leaf biochemistry Increase in chlorophyll a and b
305 G1634 OE Seed oil content Seed biochemistry Increased seed oil content
Seed protein content Decreased seed protein content
307 G1637 OE Seed protein content Seed biochemistry Altered seed protein content
309 G1640 OE Seed oil content Seed biochemistry Increased seed oil
311 G1645 OE Inflorescence Dev and morph Altered inflorescence structure
313 G1646 OE Seed oil content Seed biochemistry Increased seed oil content
315 G1652 OE Seed protein content Seed biochemistry Increased seed protein content
317 G1672 OE Seed oil content Seed biochemistry Altered seed oil content
319 G1677 OE Seed protein content Seed biochemistry Altered seed protein content
Seed oil content Seed biochemistry Altered seed oil content
321 G1749 OE Morphology: other Dev and morph Formation of necrotic lesions
323 G1750 OE Seed oil content Seed biochemistry Increased seed oil content
325 G1756 OE Botrytis Disease Increased susceptibility to Botrytis
327 G1765 OE Seed oil content Seed biochemistry Increased seed oil content
329 G1777 OE Seed oil content Seed biochemistry Increased seed oil content
Seed protein content Seed biochemistry Decreased seed protein content
331 G1792 OE Leaf Dev and morph Dark green, shiny leaves
Erysiphe Disease Increased resistance to Erysiphe
Botrytis Disease Increased resistance to Botrytis
Fusarium Disease Increased resistance to Fusarium
Nutrient uptake Abiotic stress Increased tolerance to nitrogen-
limited medium
333 G1793 OE Seed oil content Seed biochemistry Increased seed oil content
335 G1794 OE Architecture Dev and morph Altered architecture, bushier plant
Architecture Dev and morph Reduced apical dominance
Light response Dev and morph Constitutive photomorphogenesis
Osmotic Abiotic stress Increased sensitivity to high PEG
Nutrient uptake Abiotic stress Reduced root growth
337 G1804 OE Flowering time Flowering time Late flowering
Sugar sensing Sugar sensing Altered sugar sensing: more sensitive
to glucose in germination assays
339 G1818 OE Seed protein content Seed biochemistry Increased protein content
341 G1820 OE Flowering time Flowering time Early flowering
Hormone sensitivity Hormone sensitivity Reduced ABA sensitivity
Seed protein content Seed biochemistry Increased seed protein content
Osmotic Abiotic stress Better germination in high NaCl
Drought Abiotic stress Increased tolerance to drought
343 G1836 OE Sodium chloride Abiotic stress Better germination in high salt
Drought Abiotic stress Increased tolerance to drought
345 G1838 OE Seed oil content Seed biochemistry Increased seed oil content
347 G1841 OE Heat Abiotic stress Better germination under heat stress
Flowering time Flowering time Early flowering
349 G1842 OE Flowering time Flowering time Early flowering
351 G1843 OE Flowering time Flowering time Early flowering
353 G1852 OE Osmotic Abiotic stress Better root growth under osmotic stress
355 G1863 OE Leaf Dev and morph Altered leaf shape and coloration
357 G1880 KO Botrytis Disease Increased resistance to Botrytis
359 G1895 OE Flowering time Flowering time Late flowering
361 G1902 OE Seed oil content Seed biochemistry Increased seed oil content
363 G1903 OE Seed protein content Seed biochemistry Decreased seed protein content
365 G1919 OE Botrytis Disease Increased tolerance to Botrytis
367 G1927 OE Sclerotinia Disease Increased tolerance to Sclerotinia
369 G1930 OE Osmotic Abiotic stress Better germination under osmotic stress
371 G1936 KO Sclerotinia Disease Increased susceptibility to Sclerotinia
Botrytis Disease Increased susceptibility to Botrytis
373 G1944 OE Senescence Dev and morph Early senescence
375 G1946 OE Seed oil content Seed biochemistry Increased seed oil content
Seed protein content Seed biochemistry Decreased seed protein content
Flowering time Flowering time Early flowering
Nutrient uptake Abiotic stress Increased root growth on phosphate-
free media
377 G1947 KO Fertility Dev and morph Reduced fertility
379 G1948 OE Seed oil content Seed biochemistry Increased seed oil content
381 G1950 OE Botrytis Disease Increased tolerance to Botrytis
383 G1958 KO Morphology: other Dev and morph Reduced size and root mass
Seed oil content Seed biochemistry Increased seed oil content
Seed protein content Seed biochemistry Increased seed protein content.
385 G2007 OE Flowering time Flowering time Late flowering
387 G2010 OE Flowering time Flowering time Early flowering
389 G2053 OE Osmotic Abiotic stress Increased root growth under osmotic stress
391 G2059 OE Seed oil content Seed biochemistry Altered seed oil content
Seed protein content Seed biochemistry Altered seed protein content
393 G2085 OE Seed Dev and morph Increased seed size and altered seed color
395 G2105 OE Seed Dev and morph Large, pale seeds
397 G2110 OE Sodium chloride Abiotic stress Increased tolerance to high salt
399 G2114 OE Seed Dev and morph Increased seed size
401 G2117 OE Seed protein content Seed biochemistry Increased seed protein content
403 G2123 OE Seed oil content Seed biochemistry Increased seed oil content
405 G2130 OE Heat Abiotic stress Better germination in heat
407 G2133 OE Glyphosate Herbicide sensitivity Increased tolerance to glyphosate
Flowering time Flowering time Late flowering
409 G2138 OE Seed oil content Seed biochemistry Increased seed oil content
411 G2140 OE Hormone sensitivity Hormone sensitivity Decreased sensitivity to ABA
Osmotic Abiotic stress Better germination on high NaCl and
sucrose
413 G2143 OE Inflorescence Dev and morph Altered inflorescence development
Leaf Dev and morph Altered leaf shape, dark green color
Flower Dev and morph Altered flower development, ectopic
carpel tissue
415 G2144 OE Flowering time Flowering time Early flowering
Leaf Dev and morph Pale green leaves, altered leaf shape
Light response Dev and morph Long hypocotyls, altered leaf shape
417 G2153 OE Osmotic Abiotic stress Better germination under osmotic stress
419 G2155 OE Flowering time Flowering time Late flowering
421 G2192 OE Seed oil composition Seed biochemistry Altered seed fatty acid composition
423 G2295 OE Flowering time Flowering time Early flowering
425 G2340 OE Seed glucosinolates Seed biochemistry Altered glucosinolate profile
427 G2343 OE Seed oil content Seed biochemistry Increased seed oil content
429 G2346 OE Morphology: other Dev and morph Enlarged seedlings
431 G2347 OE Flowering time Flowering time Early flowering
433 G2379 OE Osmotic Abiotic stress Increased seedling vigor on high
sucrose media
435 G2430 OE Heat Abiotic stress Increased tolerance to heat
Size Dev and morph Increased leaf size, faster
development
437 G2505 OE Drought Abiotic stress Increased tolerance to drought
439 G2509 OE Seed oil content Seed biochemistry Decreased seed oil content
Seed protein content Seed biochemistry Increased seed protein content
Seed prenyl lipids Seed biochemistry Increase in alpha-tocopherol
Architecture Dev and morph Reduced apical dominance
Flowering time Flowering time Early flowering
441 G2517 OE Glyphosate Herbicide sensitivity Increased tolerance to glyphosate
443 G2520 OE Seed prenyl lipids Seed biochemistry Altered tocopherol composition
445 G2555 OE Light response Dev and morph Constitutive photomorphogenesis
Botrytis Disease Increased susceptibility to Botrytis
447 G2557 OE Leaf Dev and morph Altered leaf shape, dark green color
Flower Dev and morph Altered flower development, ectopic
carpel tissue
449 G2583 OE Leaf Dev and morph Glossy, shiny leaves
451 G2701 OE Osmotic Abiotic stress Better germination on high NaCl and
sucrose
453 G2719 OE Osmotic Abiotic stress Increased seedling vigor on high
sucrose
455 G2789 OE Osmotic Abiotic stress Better germination on high sucrose
Hormone sensitivity Hormone sensitivity Reduced ABA sensitivity
457 G2830 KO Seed oil content Seed biochemistry Increased seed oil content
1951 G12 KO Hormone sensitivity Hormone sensitivity Increased sensitivity to ACC
OE Morphology: other Dev and morph Leaf and hypocotyl necrosis
1953 G30 OE Leaf Dev and morph Glossy green leaves
Light response Dev and morph Shade avoidance
1975 G231 OE Leaf fatty acids Leaf biochemistry Increased leaf unsaturated fatty acids
Seed oil content Seed biochemistry Increased seed oil content
Seed protein content Seed biochemistry Decreased seed protein content
1979 G247 OE Trichome Dev and morph Altered trichome distribution,
reduced trichome density
1991 G370 KO Size Dev and morph Reduced size, shiny leaves
OE Trichome Dev and morph Ectropic trichome formation
2009 G485 OE Flowering time Flowering time Early flowering
KO Flowering time Flowering time Late flowering
2061 G839 OE Nutrient uptake Abiotic stress Increased tolerance to nitrogen-
limited medium
2099 G1357 OE Leaf Dev and morph Altered leaf shape, dark green leaves
Chilling Abiotic stress Increased tolerance to cold
Hormone sensitivity Hormone sensitivity Insensitive to ABA
Flowering time Flowering time Late flowering
2126 G1646 OE Seed oil content Seed oil content Increased seed oil content
2142 G1816 OE Sugar sensing Sugar sensing Increased tolerance to glucose
Nutrient uptake Abiotic stress Altered C/N sensing; less
anthocyanin
Osmotic Abiotic stress on nitrogen-limited medium
Root Dev and morph Increased tolerance to osmotic stress
Trichome Dev and morph Increased root hairs
Nutrient uptake Abiotic stress Glabrous leaves
Increased tolerance to nitrogen-
limited medium
2147 G1888 OE Size Dev and morph Reduced size, dark green leaves
2153 G1945 OE Flowering time Flowering time Late flowering
Leaf Dev and morph Altered leaf shape
2195 G2826 OE Flower Dev and morph Aerial rosettes
Trichome Dev and morph Ectropic trichome formation
2197 G2838 OE Trichome Dev and morph Increased trichome density
Flowering time Flosering time Late flowering
Flower Dev and morph Flower: multiple alterations
Flower Dev and morph Aerial rosettes
Leaves Dev and morph Dark green leaves
Size Dev and morph Increased seedling size
2199 G2839 OE Osmotic stress Dev and morph Better germination on high sucrose
Inflorescence Dev and morph Downward pedicels
Size Abiotic stress Reduced size

Table 5 shows the polypeptides identified by SEQ ID NO; Mendel Gene ID (GID) No.; the transcription factor family to which the polypeptide belongs, and conserved domains of the polypeptide. The first column shows the polypeptide SEQ ID NO; the third column shows the transcription factor family to which the polynucleotide belongs; and the fourth column shows the amino acid residue positions of the conserved domain in amino acid (AA) co-ordinates.

TABLE 5
Gene families and conserved domains
Polypeptide GID Conserved Domains in
SEQ ID NO: No. Family Amino Acid Coordinates
2 G8 AP2 151-217,243-296
4 G19 AP2 76-145
6 G22 AP2 89-157
8 G24 AP2 25-93
10 G28 AP2 145-213
12 G47 AP2 11-80
14 G156 MADS 2-57
16 G157 MADS 2-57
18 G162 MADS 2-57
20 G175 WRKY 178-234,372-428
22 G180 WRKY 118-174
24 G183 WRKY 307-363
26 G188 WRKY 175-222
28 G189 WRKY 240-297
30 G192 WRKY 128-185
32 G196 WRKY 223-283
34 G211 MYB-R1 R2R3 24-137
36 G214 MYB-related 22-71
38 G226 MYB-related 28-78
40 G241 MYB-R1 R2R3 14-114
42 G248 MYB-R1 R2R3 264-332
44 G254 MYB-related 62-106
46 G256 MYB-R1 R2R3 13-115
48 G278 AKR 2-593
50 G291 MISC 132-160
52 G303 HLH/MYC 92-161
54 G312 SCR 320-336
56 G325 Z-CO-like 5-28,48-71
58 G343 GATA/Zn 178-214
60 G353 Z-C2H2 41-61,84-104
62 G354 Z-C2H2 42-62,88-109
64 G361 Z-C2H2 43-63
66 G362 Z-C2H2 62-82
68 G371 RING/C3HC4 21-74
70 G390 HB 18-81
72 G391 HB 25-85
74 G409 HB 64-124
76 G427 HB 307-370
78 G438 HB 22-85
80 G450 IAA 6-14,78-89,112-128,180-213
82 G464 IAA 20-28,71-82,126-142,187-224
84 G470 ARF 61-393
86 G477 SBP 108-233
88 G481 CAAT 20-109
90 G482 CAAT 25-116
92 G484 CAAT 11-104
94 G489 CAAT 57-156
96 G490 CAAT 48-143
98 G504 NAC 19-174
100 G509 NAG 13-169
102 G519 NAG 11-104
104 G545 Z-C2H2 82-102,136-154
106 G546 RING/C3H2C3 114-155
108 G561 bZIP 248-308
110 G562 bZIP 253-315
112 G567 bZIP 210-270
114 G568 bZIP 215-265
116 G584 HLH/MYC 401-494
118 G585 HLH/MYC 436-501
120 G590 HLH/MYC 202-254
122 G594 HLH/MYC 140-204
124 G597 AT-hook 97-104,137-144
126 G598 DBP 205-263
128 G634 TH 62-147,189-245
130 G635 TH 239-323
132 G636 TH 55-145,405-498
134 G638 TH 119-206
136 G652 Z-CLDSH 28-49,137-151,182-196
138 G663 MYB-R1 R2R3 9-111
140 G664 MYB-R1 R2R3 13-116
142 G674 MYB-R1 R2R3 20-120
144 G676 MYB-R1 R2R3 17-119
146 G680 MYB-related 24-70
148 G682 MYB-related 27-63
150 G715 CAAT 60-132
152 G720 GARP 301-349
154 G736 Z-Dof 54-111
156 G748 Z-Dof 112-140
158 G779 HLH/MYC 126-182
160 G789 HLH/MYC 253-313
162 G801 PCF 32-93
164 G849 BPF-1 324-413,504-583
166 G859 MADS 3-56
168 G864 AP2 119-186
170 G867 AP2 59-124
172 G869 AP2 109-177
174 G877 WRKY 272-328,487-603
176 G881 WRKY 176-233
178 G892 RING/C3H2C3 177-270
180 G896 Z-LSDlike 18-39
182 G910 Z-CO-like 14-37,77-103
184 G911 RING/C3H2C3 86-129
186 G912 AP2 51-118
188 G913 AP2 62-128
190 G922 SCR 225-242
192 G926 CAAT 131-225
194 G961 NAC 15-140
196 G971 AP2 120-186
198 G974 AP2 81-140
200 G975 AP2 4-71
202 G979 AP2 63-139,165-233
204 G987 SCR 428-432,704-708
206 G988 SCR 178-195
208 G1040 GARP 109-158
210 G1047 bZIP 129-180
212 G1051 bZIP 189-250
214 G1052 bZIP 201-261
216 G1062 HLH/MYC 308-359
218 G1063 HLH/MYC 131-182
222 G1069 AT-hook 67-74
224 G1073 AT-hook 33-42,78-175
226 G1075 AT-hook 78-85
228 G1084 BZIPT2 1-53,490-619
230 G1089 BZIPT2 425-500
232 G1134 HLH/MYC 198-247
234 G1140 MADS 2-57
236 G1143 HLH/MYC 33-82
238 G1146 PAZ 886-896
240 G1196 AKR 179-254
242 G1198 bZIP 173-223
244 G1225 HLH/MYC 78-147
246 G1226 HLH/MYC 115-174
248 G1229 HLH/MYC 102-160
250 G1255 Z-CO-like 18-56
252 G1266 AP2 79-147
254 G1275 WRKY 113-169
256 G1305 MYB-R1 R2R3 15-118
258 G1322 MYB-R1 R2R3 26-130
260 G1323 MYB-R1 R2R3 15-116
262 G1330 MYB-R1 R2R3 28-134
264 G1331 MYB-R1 R2R3 8-109
266 G1332 MYB-R1 R2R3 13-116
268 G1363 CAAT 174-226
270 G1411 AP2 87-154
272 G1417 WRKY 239-296
274 G1419 AP2 69-137
276 G1449 IAA 48-53,74-107,122-152
278 G1451 ARF 22-357
280 G1452 NAC 30-177
282 G1463 NAG 9-156
284 G1471 Z-C2H2 49-70
286 G1478 Z-CO-like 32-76
288 G1482 Z-CO-like 5-63
290 G1488 GATA/Zn 221-246
292 G1494 HLH/MYC 261-311
294 G1496 HLH/MYC 184-248
296 G1499 HLH/MYC 118-181
298 G1519 RING/C3HC4 327-364
300 G1526 SWI/SNF 493-620,864-1006
302 G1540 HB 35-98
304 G1543 HB 135-195
306 G1634 MYB-related 129-180
308 G1637 MYB-related 109-173
310 G1640 MYB-R1 R2R3 14-115
312 G1645 MYB-R1 R2R3 90-210
314 G1646 CAAT 72-162
316 G1652 HLH/MYC 143-215
318 G1672 NAC 41-194
320 G1677 NAC 17-181
322 G1749 AP2 84-155
324 G1750 AP2 107-173
326 G1756 WRKY 141-197
328 G1765 NAG 20-140
330 G1777 RING/C3HC4 124-247
332 G1792 AP2 17-85
334 G1793 AP2 179-255,281-349
336 G1794 AP2 182-249
338 G1804 bZIP 357-407
340 G1818 CAAT 36-113
342 G1820 CAAT 70-133
344 G1836 CAAT 30-164
346 G1838 AP2 229-305,330-400
348 G1841 AP2 83-150
350 G1842 MADS 2-57
352 G1843 MADS 2-57
354 G1852 AKR 1-600
356 G1863 GRF-like 77-186
358 G1880 Z-C2H2 69-89,111-139
360 G1895 Z-Dof 55-110
362 G1902 Z-Dof 31-59
364 G1903 Z-Dof 134-180
398 G2110 WRKY 239-298
366 G1919 RING/C3HC4 214-287
368 G1927 NAC 17-188
370 G1930 AP2 59-124
372 G1936 PCF 64-129
374 G1944 AT-hook 87-100
376 G1946 HS 32-130
378 G1947 HS 37-120
380 G1948 AKR 75-126,120-148,152-181,
186-215,261-311,312-363
382 G1950 AKR 65-228
384 G1958 GARP 230-278
386 G2007 MYB-R1 R2R3 14-116
388 G2010 SBP 53-127
390 G2053 NAC 10-149
392 G2059 AP2 184-254
394 G2085 RING/C3HC4 214-241
396 G2105 TH 100-153
398 G2110 WRKY 239-298
400 G2114 AP2 221-297,323-393
402 G2117 bZIP 46-106
404 G2123 GF14 99-109
406 G2130 AP2 93-160
408 G2133 AP2 11-83
410 G2138 AP2 76-148
412 G2140 HLH/MYC 167-242
414 G2143 HLH/MYC 128-179
416 G2144 HLH/MYC 203-283
418 G2153 AT-hook 75-94,162-206
420 G2155 AT-hook 18-38
422 G2192 bZIP-NIN 600-700
424 G2295 MADS 2-57
426 G2340 MYB-R1 R2R3 14-120
428 G2343 MYB-R1 R2R3 14-116
430 G2346 SBP 59-135
432 G2347 SBP 60-136
434 G2379 TH 19-110,173-232
436 G2430 GARP 425-478
438 G2505 NAC 10-159
440 G2509 AP2 89-156
442 G2517 WRKY 118-174
444 G2520 HLH/MYC 135-206
446 G2555 HLH/MYC 175-245
448 G2557 HLH/MYC 278-328
450 G2583 AP2 4-71
452 G2701 MYB-related 33-81,129-183
454 G2719 MYB-R1 R2R3 56-154
456 G2789 AT-hook 53-73,121-165
458 G2830 Z-C2H2 245-266

Examples of some of the utilities that may be desirable in plants, and that may be provided by transforming the plants with the presently disclosed sequences, are listed in Table 6. Many of the transcription factors listed in Table 6 may be operably linked with a specific promoter that causes the transcription factor to be expressed in response to environmental, tissue-specific or temporal signals. For example, G362 induces ectopic trichomes on flowers but also produces small plants. The former may be desirable to produce insect or herbivore resistance, or increased cotton yield, but the latter may be undesirable in that it may reduce biomass. However, by operably linking G362 with a flower-specific promoter, one may achieve the desirable benefits of the genes without affecting overall biomass to a significant degree. For examples of flower specific promoters, see Kaiser et al. (supra). For examples of other tissue-specific, temporal-specific or inducible promoters, see the above discussion under the heading “Vectors, Promoters, and Expression Systems”.

TABLE 6
Genes, traits and utilities that affect plant characteristics
Transcription factor genes
Trait Category Phenotype(s) that impact traits Utility
Abiotic stress Effect of chilling on plants
Increased tolerance: G256; G664; G1322 Improved germination,
growth rate, earlier
planting, yield
Germination in cold
Increased tolerance: G256; G664 Earlier planting;
improved survival, yield
Freezing tolerance G720 (G720 KO is more Earlier planting;
susceptible); G912; G913 improved quality,
survival, yield
Drought
Increased tolerance: G912; G913; G1820; 61836; Improved survival,
G2505 vigor, appearance, yield
Heat
Increased tolerance: G464; G682; G864; G1305; Improved germination,
G1841; 62130; G2430 growth rate, later
planting, yield
Osmotic stress
Increased sensitivity: G1794 Abiotic stress response
manipulation
Increased tolerance: G47; G175; G188; G303; G325; Improved germination
G353; G489; G922; G926; rate, seedling vigor,
G1069; G1089; G1452; G1816; survival, yield
G1820; G1852; G1930; G2053;
G2140; G2153; G2379; G2701;
G2719; G2789; G2839
Salt tolerance
More susceptible: G545 Manipulation of
response to high salt
conditions
Increased tolerance: G22; G196; G226; G312; G482; Improved germination
G801; G867; G922; G1836; rate, survival, yield;
G2110 extended growth range
Nitrogen stress
Sensitivity to N limitation: G1794 Manipulation of
response to low nutrient
conditions
Tolerance to N limitation: G225; G226; G839; G1792; Improved yield and
G1816 nutrient stress tolerance,
decreased fertilizer
usage
Phosphate stress
Tolerance to P limitation: G545; G561; G911; G1946 Improved yield and
nutrient stress tolerance,
decreased fertilizer
usage
Oxidative stress G477 Improved yield, quality,
ultraviolet and chemical
stress tolerance
Herbicide Glyphosate G343; G2133; G2517 Generation of
glyphosate-resistant
plants to improve weed
control
Hormone Abscisic acid (ABA)
sensitivity sensitivity
Reduced sensitivity to ABA: G546; G926; G1069; G1357; Modification of seed
G1452; G1820; G2140; G2789 development, improved
seed dormancy, cold and
dehydration tolerance
Sensitivity to ethylene
Altered response: G1134 Manipulation of fruit
ripening
Insensitive to ethylene: G1330
Disease Botrytis
Increased susceptibility: G248; G371; G1064; G1084; Manipulation of
G1196; G1255; G1756; G1936; response to disease
G2555 organism
Increased resistance or G28; G1792; G1880; G1919; Improved yield,
tolerance: G1950 appearance, survival,
extended range
Fusarium
Increased susceptibility: G188; G545; G896 Manipulation of
response to disease
organism
Increased resistance or G1047; G1792 Improved yield,
tolerance: appearance, survival,
extended range
Etysiphe
Increased susceptibility: G545; G881 Manipulation of
response to disease
organism
Increased resistance or G19; G28; G409; G1266; Improved yield,
tolerance: G1363; G1792 appearance, survival,
extended range
Pseudomonas
Increased susceptibility: G545 Manipulation of
response to disease
organism
Scierotinia
Increased susceptibility: G278; G477; G594; G1936 Manipulation of
response to disease
organism
Increased resistance or G28; G1927 Improved yield,
tolerance: appearance, survival,
extended range
Growth regulator Altered sugar sensing Alteration of energy
Decreased tolerance to sugars: G241; G254; G567; G680; balance, photosynthetic
G912; G1804 rate, carbohydrate
accumulation, biomass
Increased tolerance to sugars: G481; G867; G1225; G1816 production, source-sink
relationships,
senescence; alteration of
storage compound
accumulation in seeds
Altered C/N sensing G18l6
Flowering time Early flowering G157; G180; G183; G485 (OE); Faster generation time;
G490; G590; G789; G1225; synchrony of flowering;
G1494; G1820; G1841; G1842; additional harvests
G1843; G1946; G2010; G2144; within a growing season,
G2295; G2347; G2509 shortening of breeding
programs
Late flowering G8; G47; G157; G192; G214; Increased yield or
G231; G361; G362; G485 (KO); biomass, alleviate risk of
G562; G736; G748; G859; transgenic pollen escape,
G910; G913; G971; G1051; synchrony of flowering
G1052; G1357; G1452; G1478;
G1804; G1895; G1945; G2007;
G2133; G2155; G2838
General Altered flower structure
development and Stamen: G988; G1075; G1140; G1499; Ornamental
morphology G2557 modification of plant
Sepal: G1075; G1140; G2557 architecture, improved
Petal: G638; G1075; G1140; G1449; or reduced fertility to
G1499; G2557 mitigate escape of
Pedicel: G353; G354; G988 transgenic pollen,
Carpel: G1063; G1140; G2143; G2143; improved fruit size,
G2557 shape, number or yield
Multiple alterations: G638; G988; G1063; G1140;
G1449; G1499; G2143; G2557
G988; G1449; G2838
Enlarged floral organs: G353; G354
Siliques: G470; G779; G988; G1075;
G1140; G1499; G1947; G2143;
G2557
Reduced fertility: G638; G779; G1140; G1499
Aerial rosettes G1995; G2826; G2838
Inflorescence architectural
change Ornamental
Altered branching pattern: G47; G1063; G1645; G2143 modification of flower
Short internodes/bushy G47 architecture; timing of
inflorescences: flowering; altered plant
Internode elongation: G1063 habit for yield or
Lack of inflorescence: G1499; G2143 harvestability benefit;
reduction in pollen
production of
genetically modified
plants; manipulation of
seasonality and annual
or perennial habit;
manipulation of
determinate vs.
indeterminate growth
Altered shoot meristem Ornamental
development modification of plant
Stem bifurcations: G390; G391 architecture,
manipulation of growth
and development,
increase in leaf numbers
modulation of branching
patterns to provide
improved yield or
biomass
Altered branching pattern G427; G568; G988; G1543; Ornamental
G1794 modification of plant
architecture, improved
lodging resistance
Apical dominance Ornamental
Reduced apical dominance: G47; G211; G1255; G1275; modification of plant
G1411; G1488; G1794; G2509 architecture
Altered trichome density; Ornamental
development, or structure modification of plant
architecture, increased
Reduced or no trichomes: G225; G226; G247; G585; plant product (e.g.,
G676; G682; G1332; G1452; diterpenes, cotton)
G1816 productivity, insect and
herbivore resistance
Ectopic trichomes/altered G247; G362; G370; G676;
trichome development/cell G2826
fate:
Increase in trichome number, G362; G634; G838; G2838
size or density:
Stem morphology and altered G47; G438; G748; G988; Modulation of lignin
vascular tissue structure G1488 content; improvement of
wood, palatability of
fruits and vegetables
Root development
Increased root growth and G1482
proliferation: Improved yield, stress
tolerance; anchorage
Increased root hairs: G225; G226; G1816
Altered seed development, G979
ripening and germination
Cell differentiation and cell G1540 Increase in carpel or
proliferation fruit development;
Improve regeneration of
shoots from callus in
transformation or micro-
propagation systems
Rapid development G2430 Promote faster
development and
reproduction in plants
Senescence
Premature senescence: G636; G1463; G1944 Improvement in
response to disease, fruit
ripening
Lethality when overexpressed G877; G1519 Herbicide target;
ablation of specific
tissues or organs such as
stamen to prevent pollen
escape
Necrosis G12, G24 Disease resistance
Plant size Increased plant size G1073; G1451 Improved yield,
biomass, appearance
Larger seedlings G2346; G2838 Increased survival and
vigor of seedlings, yield
Dwarfed or more compact G24; G343; G353; G354; G362; Dwarfism, lodging
plants G370; G1008; G1277; G1543; resistance, manipulation
G1794; G1958 of gibberellin responses
Leaf morphology Dark green leaves G674; G912; G1063; G1357; Increased
G1452; G1482; G1499; G1792; photosynthesis, biomass,
G1863; G1888; G2143; G2557; appearance, yield
G2838
Change in leaf shape G211; G353; G674; G736; Ornamental applications
G1063; G1146; G1357; G1452;
G1494; G1543; G1863; G2143;
G2144
Altered leaf size:
Increased leaf size, number or G189; G214; G1451; G2430 Increased yield,
mass: ornamental applications
Light green leaves G1494; G2144 Ornamental applications
Variegation G635 Ornamental applications
Glossy leaves G30; G1792; G2583 Ornamental
applications,
manipulation of wax
composition, amount, or
distribution
Seed morphology Altered seed coloration G156; G2105; G2085 Appearance
Seed size and shape
Increased seed size: G450; G584; G1255; G2085; Yield, appearance
G2105; G2114
Decreased seed size: G01040 Appearance
Altered seed shape: G1040; G1062 Appearance
Leaf biochemistry Increased leaf wax G975; G1792; G2583 Insect, pathogen
resistance
Leaf prenyl lipids
Reduced chlorophyll: G987
Increase in tocopherols G652; G987; G2509
Increased lutein content G748
Increase in chlorophyll or G214; G1543
carotenoids:
Leaf insoluble sugars
Increase in leaf xylose G211
Increased leaf anthocyanins G663; G1482; G1888
Leaf fatty acids
Reduction in leaf fatty acids: G987
Increase in leaf fatty acids: G214
Seed Seed oil content
biochemistry Increased oil content: G162; G291; G427; G509; Improved oil yield
G519; G561; G590; G598;
G629; G715; G849; G961;
G1198; G1226; G1471; G1478;
G1526; G1640; G1646; G1750; Reduced caloric content
G1765; G1777; G1793; G1838;
Gl902; G1946; G1948; G1958,
G2123; G2138; G2343; G2830
Decreased oil content: G180; G192; G241; G504;
G1143; G1229; G1323; G1543;
G2509
Altered oil content: G567; G892; G974; G1451;
G1496; G1646; G1672; G1677
Altered fatty acid content: G869; G1417; G2192
Seed protein content
Increased protein content: G162; G226; G241; G509; Improved protein yield,
G988; G1323; G1419; G1652; nutritional value
G1818; G1820; G1958; G2117;
G2509 Reduced caloric content
Decreased protein content: G427; G1478; G1777; G1903;
G1946
Altered protein content: G162; G567; G597; G849;
G892; G1634; G1637; G1677
Altered seed prenyl lipid G652; G2509; G2520 Improved antioxidant
content or composition and vitamin B content
Seed glucosinolate
Altered profile: G484; G2340
Increased seed anthocyanins G362; G663
Root Increased root anthocyanins G663
Biochemistry
Light Altered cotyledon, hypocotyl, G183; G354; G1322; G1331; Potential for increased
response/shade petiole development; altered G1488; G1494; G1794; G2144; planting densities and
avoidance leaf orientation; constitutive G2555 yield enhancement
photomorphogenesis;
photomorphogenesis in low
light
Pigment Increased anthocyanin level G362; G663; G1482 Enhanced health
benefits, improved
ornamental appearance,
increased stress
resistance, attraction of
pollinating and seed
dispersing animals
Abbreviations:
N = nitrogen
P = phosphate
ABA = abscisic acid
C/N = carbon/nitrogen balance

Detailed Description of Genes, Traits and Utilities that Affect Plant Characteristics

The following descriptions of traits and utilities associated with the present transcription factors offer a more comprehensive description than that provided in Table 6.

Abiotic Stress, General Considerations

Plant transcription factors can modulate gene expression, and, in turn, be modulated by the environmental experience of a plant. Significant alterations in a plant's environment invariably result in a change in the plant's transcription factor gene expression pattern. Altered transcription factor expression patterns generally result in phenotypic changes in the plant. Transcription factor gene product(s) in transgenic plants then differ(s) in amounts or proportions from that found in wild-type or non-transformed plants, and those transcription factors likely represent polypeptides that are used to alter the response to the environmental change. By way of example, it is well accepted in the art that analytical methods based on altered expression patterns may be used to screen for phenotypic changes in a plant far more effectively than can be achieved using traditional methods.

Abiotic stress: adult stage chilling. Enhanced chilling tolerance may extend the effective growth range of chilling sensitive crop species by allowing earlier planting or later harvest. Improved chilling tolerance may be conferred by increased expression of glycerol-3-phosphate acetyltransferase in chloroplasts (see, for example, Wolter et al. (1992) et al. EMBO J. 4685-4692, and Murata et al. (1992) Nature 356: 710-713).

Chilling tolerance could also serve as a model for understanding how plants adapt to water deficit. Both chilling and water stress share similar signal transduction pathways and tolerance/adaptation mechanisms. For example, acclimation to chilling temperatures can be induced by water stress or treatment with abscisic acid. Genes induced by low temperature include dehydrins (or LEA proteins). Dehydrins are also induced by salinity, abscisic acid, water stress, and during the late stages of embryogenesis.

Another large impact of chilling occurs during post-harvest storage. For example, some fruits and vegetables do not store well at low temperatures (for example, bananas, avocados, melons, and tomatoes). The normal ripening process of the tomato is impaired if it is exposed to cool temperatures. Transcription factor genes conferring resistance to chilling temperatures, including G256, G664, and G1322 may thus enhance tolerance during post-harvest storage.

Abiotic stress: cold germination. Several of the presently disclosed transcription factor genes confer better germination and growth in cold conditions. For example, the improved germination in cold conditions seen with G256 and G664 indicates a role in regulation of cold responses by these genes and their equivalogs. These genes might be engineered to manipulate the response to low temperature stress. Genes that would allow germination and seedling vigor in the cold would have highly significant utility in allowing seeds to be planted earlier in the season with a high rate of survival. Transcription factor genes that confer better survival in cooler climates allow a grower to move up planting time in the spring and extend the growing season further into autumn for higher crop yields. Germination of seeds and survival at temperatures significantly below that of the mean temperature required for germination of seeds and survival of non-transformed plants would increase the potential range of a crop plant into regions in which it would otherwise fail to thrive.

Abiotic stress: freezing tolerance and osmotic stress. Presently disclosed transcription factor genes, including G47, G175, G188, G303, G325, G353, G489, G922, G926, G1069, G1089, G1452, G1820, G1852, G1930, G2053, G2140, G2153, G2379, G2701, G2719, G2789, G2839 and their equivalogs, that increase germination rate and/or growth under adverse osmotic conditions, could impact survival and yield of seeds and plants. Osmotic stresses may be regulated by specific molecular control mechanisms that include genes controlling water and ion movements, functional and structural stress-induced proteins, signal perception and transduction, and free radical scavenging, and many others (Wang et al. (2001) Acta Hort. (ISHS) 560: 285-292). Instigators of osmotic stress include freezing, drought and high salinity, each of which are discussed in more detail below.

In many ways, freezing, high salt and drought have similar effects on plants, not the least of which is the induction of common polypeptides that respond to these different stresses. For example, freezing is similar to water deficit in that freezing reduces the amount of water available to a plant. Exposure to freezing temperatures may lead to cellular dehydration as water leaves cells and forms ice crystals in intercellular spaces (Buchanan, supra). As with high salt concentration and freezing, the problems for plants caused by low water availability include mechanical stresses caused by the withdrawal of cellular water. Thus, the incorporation of transcription factors that modify a plant's response to osmotic stress or improve tolerance to (e.g., by G720, G912, G913 or their equivalogs) into, for example, a crop or ornamental plant, may be useful in reducing damage or loss. Specific effects caused by freezing, high salt and drought are addressed below.

Abiotic stress: drought and low humidity tolerance. Exposure to dehydration invokes similar survival strategies in plants as does freezing stress (see, for example, Yelenosky (1989) Plant Physiol 89: 444-451) and drought stress induces freezing tolerance (see, for example, Siminovitch et al. (1982) Plant Physiol 69: 250-255; and Guy et al. (1992) Planta 188:265-270). In addition to the induction of cold-acclimation proteins, strategies that allow plants to survive in low water conditions may include, for example, reduced surface area, or surface oil or wax production. A number of presently disclosed transcription factor genes, e.g., G912, G913, G1820, G1836 and G2505 increase a plant's tolerance to low water conditions and, along with their functional equivalogs, would provide the benefits of improved survival, increased yield and an extended geographic and temporal planting range.

Abiotic stress: heat stress tolerance. The germination of many crops is also sensitive to high temperatures. Presently disclosed transcription factor genes that provide increased heat tolerance, including G464, G682, G864, G1305, G1841, G2130, G2430 and their equivalogs, would be generally useful in producing plants that germinate and grow in hot conditions, may find particular use for crops that are planted late in the season, or extend the range of a plant by allowing growth in relatively hot climates.

Abiotic stress: salt. The genes in Table 6 that provide tolerance to salt may be used to engineer salt tolerant crops and trees that can flourish in soils with high saline content or under drought conditions. In particular, increased salt tolerance during the germination stage of a plant enhances survival and yield. Presently disclosed transcription factor genes, including G22, G1196, G226, G312, G482, G801, G867, G922, G1836, G2110, and their equivalogs that provide increased salt tolerance during germination, the seedling stage, and throughout a plant's life cycle, would find particular value for imparting survival and yield in areas where a particular crop would not normally prosper.

Nutrient uptake and utilization: nitrogen and phosphorus. Presently disclosed transcription factor genes introduced into plants provide a means to improve uptake of essential nutrients, including nitrogenous compounds, phosphates, potassium, and trace minerals. The enhanced performance of, for example, G225, G226, G839, G1792, and other overexpressing lines under low nitrogen, and G545, G561, G911, G1946 under low phosphorous conditions indicate that these genes and their equivalogs can be used to engineer crops that could thrive under conditions of reduced nutrient availability. Phosphorus, in particular, tends to be a limiting nutrient in soils and is generally added as a component in fertilizers. Young plants have a rapid intake of phosphate and sufficient phosphate is important for yield of root crops such as carrot, potato and parsnip.

The effect of these modifications is to increase the seedling germination and range of ornamental and crop plants. The utilities of presently disclosed transcription factor genes conferring tolerance to conditions of low nutrients also include cost savings to the grower by reducing the amounts of fertilizer needed, environmental benefits of reduced fertilizer runoff into watersheds; and improved yield and stress tolerance. In addition, by providing improved nitrogen uptake capability, these genes can be used to alter seed protein amounts and/or composition in such a way that could impact yield as well as the nutritional value and production of various food products.

A number of the transcription factor-overexpressing lines make less anthocyanin on high sucrose plus glutamine indicates that these genes can be used to modify carbon and nitrogen status, and hence assimilate partitioning (assimilate partitioning refers to the manner in which an essential element, such as nitrogen, is distributed among different pools inside a plant, generally in a reduced form, for the purpose of transport to various tissues).

Increased tolerance of plants to oxidative stress. In plants, as in all living things, abiotic and biotic stresses induce the formation of oxygen radicals, including superoxide and peroxide radicals. This has the effect of accelerating senescence, particularly in leaves, with the resulting loss of yield and adverse effect on appearance. Generally, plants that have the highest level of defense mechanisms, such as, for example, polyunsaturated moieties of membrane lipids, are most likely to thrive under conditions that introduce oxidative stress (e.g., high light, ozone, water deficit, particularly in combination). Introduction of the presently disclosed transcription factor genes, including G477 and its equivalogs, that increase the level of oxidative stress defense mechanisms would provide beneficial effects on the yield and appearance of plants. One specific oxidizing agent, ozone, has been shown to cause significant foliar injury, which impacts yield and appearance of crop and ornamental plants. In addition to reduced foliar injury that would be found in ozone resistant plant created by transforming plants with some of the presently disclosed transcription factor genes, the latter have also been shown to have increased chlorophyll fluorescence (Yu-Sen Changet al. (2001) Bot. Bull. Acad. Sin. 42: 265-272).

Decreased herbicide sensitivity. Presently disclosed transcription factor genes, including G343, G2133, G2517 and their equivalogs, that confer resistance or tolerance to herbicides (e.g., glyphosate) will find use in providing means to increase herbicide applications without detriment to desirable plants. This would allow for the increased use of a particular herbicide in a local environment, with the effect of increased detriment to undesirable species and less harm to transgenic, desirable cultivars.

Knockouts of a number of the presently disclosed transcription factor genes have been shown to be lethal to developing embryos. Thus, these genes are potentially useful as herbicide targets.

Hormone sensitivity. ABA plays regulatory roles in a host of physiological processes in all higher as well as in lower plants (Davies et al. (1991) Abscisic Acid: Physiology and Biochemistry. Bios Scientific Publishers, Oxford, UK; Zeevaart et al. (1988) Ann Rev Plant Physiol. Plant Mol. Biol. 49: 439-473; Shimizu-Sato et al. (2001) Plant Physiol 127: 1405-1413). ABA mediates stress tolerance responses in higher plants, is a key signal compound that regulates stomatal aperture and, in concert with other plant signaling compounds, is implicated in mediating responses to pathogens and wounding or oxidative damage (for example, see Larkindale et al. (2002) Plant Physiol. 128: 682-695). In seeds, ABA promotes seed development, embryo maturation, synthesis of storage products (proteins and lipids), desiccation tolerance, and is involved in maintenance of dormancy (inhibition of germination), and apoptosis (Zeevaart et al. (1988) Ann Rev Plant Physiol. Plant Mol. Biol. 49: 439-473; Davies (1991), supra; Thomas (1993) Plant Cell 5: 1401-1410; and Bethke et al. (1999) Plant Cell 11: 1033-1046). ABA also affects plant architecture, including root growth and morphology and root-to-shoot ratios. ABA action and metabolism is modulated not only by environmental signals but also by endogenous signals generated by metabolic feedback, transport, hormonal cross-talk and developmental stage. Manipulation of ABA levels, and hence by extension the sensitivity to ABA, has been described as a very promising means to improve productivity, performance and architecture in plants Zeevaart (1999) in: Biochemistry and Molecular Biology of Plant Hormones, Hooykaas et al. eds, Elsevier Science pp 189-207; and Cutler et al. (1999) Trends Plant Sci. 4: 472-478).

A number of the presently disclosed transcription factor genes affect plant abscisic acid (ABA) sensitivity, including G546, G926, 1069, G1357, G1452, G1820, G2140, G2789. Thus, by affecting ABA sensitivity, these introduced transcription factor genes and their equivalogs would affect cold, drought, oxidative and other stress sensitivities, plant architecture, and yield.

Several other of the present transcription factor genes have been used to manipulate ethylene signal transduction and response pathways. These genes can thus be used to manipulate the processes influenced by ethylene, such as seed germination or fruit ripening, and to improve seed or fruit quality.

Diseases, pathogens and pests. A number of the presently disclosed transcription factor genes have been shown to or are likely to affect a plants response to various plant diseases, pathogens and pests. The offending organisms include fungal pathogens Fusarium oxysporum, Botrytis cinerea, Sclerotinia sclerotiorum, and Erysiphe orontii. Bacterial pathogens to which resistance may be conferred include Pseudomonas syringae. Other problem organisms may potentially include nematodes, mollicutes, parasites, or herbivorous arthropods. In each case, one or more transformed transcription factor genes may provide some benefit to the plant to help prevent or overcome infestation, or be used to manipulate any of the various plant responses to disease. These mechanisms by which the transcription factors work could include increasing surface waxes or oils, surface thickness, or the activation of signal transduction pathways that regulate plant defense in response to attacks by herbivorous pests (including, for example, protease inhibitors). Another means to combat fungal and other pathogens is by accelerating local cell death or senescence, mechanisms used to impair the spread of pathogenic microorganisms throughout a plant. For instance, the best known example of accelerated cell death is the resistance gene-mediated hypersensitive response, which causes localized cell death at an infection site and initiates a systemic defense response. Because many defenses, signaling molecules, and signal transduction pathways are common to defense against different pathogens and pests, such as fungal, bacterial, oomycete, nematode, and insect, transcription factors that are implicated in defense responses against the fungal pathogens tested may also function in defense against other pathogens and pests. These transcription factors include, for example, G28, G1792, G1880, G1919, G1950 (improved resistance or tolerance to Botrytis), G1047, G1792 (improved resistance or tolerance to Fusarium), G19, G28, G409, G1266, G1363, G1792 (improved resistance or tolerance to Erysiphe), G545 (improved resistance or tolerance to Pseudomonas), G28, G1927 (improved resistance or tolerance to Sclerotinia), and their equivalogs.

Growth regulator: sugar sensing. In addition to their important role as an energy source and structural component of the plant cell, sugars are central regulatory molecules that control several aspects of plant physiology, metabolism and development (Hsieh et al. (1998) Proc. Natl. Acad. Sci. 95: 13965-13970). It is thought that this control is achieved by regulating gene expression and, in higher plants, sugars have been shown to repress or activate plant genes involved in many essential processes such as photosynthesis, glyoxylate metabolism, respiration, starch and sucrose synthesis and degradation, pathogen response, wounding response, cell cycle regulation, pigmentation, flowering and senescence. The mechanisms by which sugars control gene expression are not understood.

Because sugars are important signaling molecules, the ability to control either the concentration of a signaling sugar or how the plant perceives or responds to a signaling sugar could be used to control plant development, physiology or metabolism. For example, the flux of sucrose (a disaccharide sugar used for systemically transporting carbon and energy in most plants) has been shown to affect gene expression and alter storage compound accumulation in seeds. Manipulation of the sucrose signaling pathway in seeds may therefore cause seeds to have more protein, oil or carbohydrate, depending on the type of manipulation. Similarly, in tubers, sucrose is converted to starch which is used as an energy store. It is thought that sugar signaling pathways may partially determine the levels of starch synthesized in the tubers. The manipulation of sugar signaling in tubers could lead to tubers with a higher starch content.

Thus, the presently disclosed transcription factor genes that manipulate the sugar signal transduction pathway, including G241, G254, G567, G680, G912, G1804, G481, G867, G1225, along with their equivalogs, may lead to altered gene expression to produce plants with desirable traits. In particular, manipulation of sugar signal transduction pathways could be used to alter source-sink relationships in seeds, tubers, roots and other storage organs leading to increase in yield.

Growth regulator: C/N sensing. Nitrogen and carbon metabolism are tightly linked in almost every biochemical pathway in the plant. Carbon metabolites regulate genes involved in N acquisition and metabolism, and are known to affect germination and the expression of photosynthetic genes (Coruzzi et al. (2001) Plant Physiol. 125: 61-64) and hence growth. Early studies on nitrate reductase (NR) in 1976 showed that NR activity could be affected by Glc/Suc (Crawford (1995) Plant Cell 7: 859-886; Daniel-Vedele et al. (1996) CR Acad Sci Paris 319: 961-968). Those observations were supported by later experiments that showed sugars induce NR mRNA in dark-adapted, green seedlings (Cheng CL, et al. (1992) Proc Natl Acad Sci USA 89: 1861-1864). C and N may have antagonistic relationships as signaling molecules; light induction of NR activity and mRNA levels can be mimicked by C metabolites and N-metabolites cause repression of NR induction in tobacco (Vincentz et al. (1992) Plant J 3: 315-324). Gene regulation by C/N status has been demonstrated for a number of N-metabolic genes (Stitt (1999) Curr. Opin. Plant. Biol. 2: 178-186); Coruzzi et al. (2001) supra). Thus, transcription factor genes that affect C/N sensing, such as G1816, can be used to alter or improve germination and growth under nitrogen-limiting conditions.

Flowering time: early and late flowering. Presently disclosed transcription factor genes that accelerate flowering, which include G157, G180, G183, G485, G490, G590, G789, G1225, G1494, G1820, G1841, G1842, G1843, G1946, G2010, G2144, G2295, G2347, G2509, and their functional equivalogs, could have valuable applications in such programs, since they allow much faster generation times. In a number of species, for example, broccoli, cauliflower, where the reproductive parts of the plants constitute the crop and the vegetative tissues are discarded, it would be advantageous to accelerate time to flowering. Accelerating flowering could shorten crop and tree breeding programs. Additionally, in some instances, a faster generation time would allow additional harvests of a crop to be made within a given growing season. A number of Arabidopsis genes have already been shown to accelerate flowering when constitutively expressed. These include LEAFY, APETALA1 and CONSTANS (Mandel et al. (1995) Nature 377: 522-524; Weigel and Nilsson (1995) Nature 377:et al. 495-500; Simon et al. (1996) Nature 384: 59-62).

By regulating the expression of potential flowering using inducible promoters, flowering could be triggered by application of an inducer chemical. This would allow flowering to be synchronized across a crop and facilitate more efficient harvesting. Such inducible systems could also be used to tune the flowering of crop varieties to different latitudes. At present, species such as soybean and cotton are available as a series of maturity groups that are suitable for different latitudes on the basis of their flowering time (which is governed by day-length). A system in which flowering could be chemically controlled would allow a single high-yielding northern maturity group to be grown at any latitude. In southern regions such plants could be grown for longer periods before flowering was induced, thereby increasing yields. In more northern areas, the induction would be used to ensure that the crop flowers prior to the first winter frosts.

In a sizeable number of species, for example, root crops, where the vegetative parts of the plants constitute the crop and the reproductive tissues are discarded, it is advantageous to identify and incorporate transcription factor genes that delay or prevent flowering in order to prevent resources being diverted into reproductive development. For example, G8, G47, G157, G192, G214, G231; G361, G362 , G562, G736, G748, G859, G910, G913, G971, G1051, G1052, G1357, G1452, G1478, G1804, G1895, G1945, G2007, G2133, G2155, G2838 and equivalogs, delay flowering time in transgenic plants. Extending vegetative development with presently disclosed transcription factor genes could thus bring about large increases in yields. Prevention of flowering can help maximize vegetative yields and prevent escape of genetically modified organism (GMO) pollen.

Presently disclosed transcription factors that extend flowering time have utility in engineering plants with longer-lasting flowers for the horticulture industry, and for extending the time in which the plant is fertile.

A number of the presently disclosed transcription factors may extend flowering time, and delay flower abscission, which would have utility in engineering plants with longer-lasting flowers for the horticulture industry. This would provide a significant benefit to the ornamental industry, for both cut flowers and woody plant varieties (of, for example, maize), as well as have the potential to lengthen the fertile period of a plant, which could positively impact yield and breeding programs.

General development and morphology: flower structure and inflorescence: architecture, altered flower organs, reduced fertilitv, multiple alterations, aerial rosettes, branching, internode distance, terminal flowers and phase change. Presently disclosed transgenic transcription factors such as G353; G354, G638; G779; G988; G1063; G1075; G1140; G1449; G1499; G2143; 62557, G2838, G2839 and their equivalogs, may be used to create plants with larger flowers or arrangements of flowers that are distinct from wild-type or non-transformed cultivars. This would likely have the most value for the ornamental horticulture industry, where larger flowers or interesting floral configurations are generally preferred and command the highest prices.

Flower structure may have advantageous or deleterious effects on fertility, and could be used, for example, to decrease fertility by the absence, reduction or screening of reproductive components. In fact, plants that overexpress a sizable number of the presently disclosed transcription factor genes e.g., G470, G779, G988, G1075, G1140, G1499, G1947, G2143, G2557 and their functional equivalogs, possess reduced fertility; flowers are infertile and fail to yield seed. These could be desirable traits, as low fertility could be exploited to prevent or minimize the escape of the pollen of genetically modified organisms (GMOs) into the environment.

The alterations in shoot architecture seen in the lines transformed with G47, G1063, G1645, G2143, and their functional equivalogs indicates that these genes and their equivalogs can be used to manipulate inflorescence branching patterns. This could influence yield and offer the potential for more effective harvesting techniques. For example, a “self pruning” mutation of tomato results in a determinate growth pattern and facilitates mechanical harvesting (Pnueli et al. (2001) Plant Cell 13(12): 2687-702).

One interesting application for manipulation of flower structure, for example, by introduced transcription factors could be in the increased production of edible flowers or flower parts, including saffron, which is derived from the stigmas of Crocus sativus.

Genes that later silique conformation in brassicates may be used to modify fruit ripening processes in brassicates and other plants, which may positively affect seed or fruit quality.

A number of the presently disclosed transcription factors may affect the timing of phase changes in plants. Since the timing or phase changes generally affects a plant's eventual size, these genes may prove beneficial by providing means for improving yield and biomass.

General development and morphology: shoot meristem and branching patterns. Several of the presently disclosed transcription factor genes, including G390 and G391, and G1794, when introduced into plants, have been shown to cause stem bifurcations in developing shoots in which the shoot meristems split to form two or three separate shoots. These transcription factors and their functional equivalogs may thus be used to manipulate branching. This would provide a unique appearance, which may be desirable in ornamental applications, and may be used to modify lateral branching for use in the forestry industry. A reduction in the formation of lateral branches could reduce knot formation. Conversely, increasing the number of lateral branches could provide utility when a plant is used as a view- or windscreen.

General development and morphology: apical dominance: The modified expression of presently disclosed transcription factors (e.g., G47, G211, G1255, G1275, G1411, G1488, G1794, G2509 and their equivalogs) that reduce apical dominance could be used in ornamental horticulture, for example, to modify plant architecture, for example, to produce a shorter, more bushy stature than wild type. The latter form would have ornamental utility as well as provide increased resistance to lodging.

General development and morphology: trichome density, development or structure. Several of the presently disclosed transcription factor genes have been used to modify trichome number, density, trichome cell fate, amount of trichome products produced by plants, or produce ectopic trichome formation. These include G225; G226, G247; G362 , G370; G585, G634, G676, G682, G1332, G1452, G1995, G2826, and G2838. In most cases where the metabolic pathways are impossible to engineer, increasing trichome density or size on leaves may be the only way to increase plant productivity. Thus, by increasing trichome density, size or type, these trichome-affecting genes and their functional equivalogs would have profound utilities in molecular farming practices by making use of trichomes as a manufacturing system for complex secondary metabolites.

Trichome glands on the surface of many higher plants produce and secrete exudates that give protection from the elements and pests such as insects, microbes and herbivores. These exudates may physically immobilize insects and spores, may be insecticidal or ant-microbial or they may act as allergens or irritants to protect against herbivores. By modifying trichome location, density or activity with presently disclosed transcription factors that modify these plant characteristics, plants that are better protected and higher yielding may be the result.

A potential application for these trichome-affecting genes and their equivalogs also exists in cotton: cotton fibers are modified unicellular trichomes that develop from the outer ovule epidermis. In fact, only about 30% of these epidermal cells develop into trichomes, but all have the potential to develop a trichome fate. Trichome-affecting genes can trigger an increased number of these cells to develop as trichomes and thereby increase the yield of cotton fibers. Since the mallow family is closely related to the Brassica family, genes involved in trichome formation will likely have homologs in cotton or function in cotton.

If the effects on trichome patterning reflect a general change in heterochronic processes, trichome-affecting transcription factors or their equivalogs can be used to modify the way meristems and/or cells develop during different phases of the plant life cycle. In particular, altering the timing of phase changes could afford positive effects on yield and biomass production.

General development and morphology: stem morphologv and altered vascular tissue structure. Plants transformed with transcription factor genes that modify stem morphology or lignin content may be used to affect overall plant architecture and the distribution of lignified fiber cells within the stem.

Modulating lignin content might allow the quality of wood used for furniture or construction to be improved. Lignin is energy rich; increasing lignin composition could therefore be valuable in raising the energy content of wood used for fuel. Conversely, the pulp and paper industries seek wood with a reduced lignin content. Currently, lignin must be removed in a costly process that involves the use of many polluting chemicals. Consequently, lignin is a serious barrier to efficient pulp and paper production (Tzfira et al. (1998) TIBTECH 16: 439-446; Robinson (1999) Nature Biotechnology 17: 27-30). In addition to forest biotechnology applications, changing lignin content by selectively expressing or repressing transcription factors in fruits and vegetables might increase their palatability.

Transcription factors that modify stem structure, including G47, G438, G748, G988, G1488 and their equivalogs, may also be used to achieve reduction of higher-order shoot development, resulting in significant plant architecture modification. Overexpression of the genes that encode these transcription factors in woody plants might result in trees that lack side branches, and have fewer knots in the wood. Altering branching patterns could also have applications amongst ornamental and agricultural crops. For example, applications might exist in any species where secondary shoots currently have to be removed manually, or where changes in branching pattern could increase yield or facilitate more efficient harvesting.

General development and morphology: altered root development. By modifying the structure or development of roots by transforming into a plant one or more of the presently disclosed transcription factor genes, including G225, G226, G1482, and their equivalogs, plants may be produced that have the capacity to thrive in otherwise unproductive soils. For example, grape roots extending further into rocky soils would provide greater anchorage, greater coverage with increased branching, or would remain viable in waterlogged soils, thus increasing the effective planting range of the crop and/or increasing yield and survival. It may be advantageous to manipulate a plant to produce short roots, as when a soil in which the plant will be growing is occasionally flooded, or when pathogenic fungi or disease-causing nematodes are prevalent.

General development and morphology: seed development, ripening and germination rate. A number of the presently disclosed transcription factor genes (e.g., G979) have been shown to modify seed development and germination rate, including when the seeds are in conditions normally unfavorable for germination (e.g., cold, heat or salt stress, or in the presence of ABA), and may, along with functional equivalogs, thus be used to modify and improve germination rates under adverse conditions.

General development and morphology: cell differentiation and cell proliferation. Several of the disclosed transcription factors regulate cell proliferation and/or differentiation, including G1540 and its functional equivalogs. Control of these processes could have valuable applications in plant transformation, cell culture or micro-propagation systems, as well as in control of the proliferation of particular useful tissues or cell types. Transcription factors that induce the proliferation of undifferentiated cells can be operably linked with an inducible promoter to promote the formation of callus that can be used for transformation or production of cell suspension cultures. Transcription factors that prevent cells from differentiating, such as G1540 or its equivalogs, could be used to confer stem cell identity to cultured cells. Transcription factors that promote differentiation of shoots could be used in transformation or micro-propagation systems, where regeneration of shoots from callus is currently problematic. In addition, transcription factors that regulate the differentiation of specific tissues could be used to increase the proportion of these tissues in a plant. Genes that promote the differentiation of carpet tissue could be introduced into commercial species to induce formation of increased numbers of carpets or fruits. A particular application might exist in saffron, one of the world's most expensive spices. Saffron filaments, or threads, are actually the dried stigmas of the saffron flower, Crocus sativus Linneaus. Each flower contains only three stigmas, and more than 75,000 of these flowers are needed to produce just one pound of saffron filaments. An increase in carpel number would increase the quantity of stigmatic tissue and improve yield.

General development and morphology: cell expansion. Plant growth results from a combination of cell division and cell expansion. Transcription factors may be useful in regulation of cell expansion. Altered regulation of cell expansion could affect stem length, an important agronomic characteristic. For instance, short cultivars of wheat contributed to the Green Revolution, because plants that put fewer resources into stem elongation allocate more resources into developing seed and produce higher yield. These plants are also less vulnerable to wind and rain damage. These cultivars were found to be altered in their sensitivity to gibberellins, hormones that regulate stem elongation through control of both cell expansion and cell division. Altered cell expansion in leaves could also produce novel and ornamental plant forms.

General development and morphology: phase change and floral reversion. Transcription factors that regulate phase change can modulate the developmental programs of plants and regulate developmental plasticity of the shoot meristem. In particular, these genes might be used to manipulate seasonality and influence whether plants display an annual or perennial habit.

General development and morphology: rapid development. A number of the presently disclosed transcription factor genes, including G2430, have been shown to have significant effects on plant growth rate and development. These observations have included, for example, more rapid or delayed growth and development of reproductive organs. Thus, by causing more rapid development, G2430 and its functional equivalogs would prove useful for regions with short growing seasons; other transcription factors that delay development may be useful for regions with longer growing seasons. Accelerating plant growth would also improve early yield or increase biomass at an earlier stage, when such is desirable (for example, in producing forestry products or vegetable sprouts for consumption). Transcription factors that promote faster development such as G2430 and its functional equivalogs may also be used to modify the reproductive cycle of plants.

General development and morphology: slow growth rate. A number of the presently disclosed transcription factor genes, including G652 and G1335, have been shown to have significant effects on retarding plant growth rate and development. These observations have included, for example, delayed growth and development of reproductive organs. Slow growing plants may be highly desirable to ornamental horticulturists, both for providing house plants that display little change in their appearance over time, or outdoor plants for which wild-type or rapid growth is undesirable (e.g., ornamental palm trees). Slow growth may also provide for a prolonged fruiting period, thus extending the harvesting season, particularly in regions with long growing seasons. Slow growth could also provide a prolonged period in which pollen is available for improved self- or cross-fertilization, or cross-fertilization of cultivars that normally flower over non-overlapping time periods. The latter aspect may be particularly useful to plants comprising two or more distinct grafted cultivars (e.g., fruit trees) with normally non-overlapping flowering periods.

General development and morphology: senescence. Presently disclosed transcription factor genes may be used to alter senescence responses in plants. Although leaf senescence is thought to be an evolutionary adaptation to recycle nutrients, the ability to control senescence in an agricultural setting has significant value. For example, a delay in leaf senescence in some maize hybrids is associated with a significant increase in yields and a delay of a few days in the senescence of soybean plants can have a large impact on yield. In an experimental setting, tobacco plants engineered to inhibit leaf senescence had a longer photosynthetic lifespan, and produced a 50% increase in dry weight and seed yield (Gan and Amasino (1995) Science 270: 1986-1988). Delayed flower senescence caused by overexpression of transcription factors may generate plants that retain their blossoms longer and this may be of potential interest to the ornamental horticulture industry, and delayed foliar and fruit senescence could improve post-harvest shelf-life of produce.

Premature senescence caused by, for example, G636, G1463, G1944 and their equivalogs may be used to improve a plant's response to disease and hasten fruit ripening.

Growth rate and development: lethality and necrosis. Overexpression of transcription factors, for example, G12, G24, G877, G1519 and their equivalogs that have a role in regulating cell death may be used to induce lethality in specific tissues or necrosis in response to pathogen attack. For example, if a transcription factor gene inducing lethality or necrosis was specifically active in gametes or reproductive organs, its expression in these tissues would lead to ablation and subsequent male or female sterility. Alternatively, under pathogen-regulated expression, a necrosis-inducing transcription factor can restrict the spread of a pathogen infection through a plant.

Plant size: large plants. Plants overexpressing G1073 and G1451, for example, have been shown to be larger than controls. For some ornamental plants, the ability to provide larger varieties with these genes or their equivalogs may be highly desirable. For many plants, including fruit-bearing trees, trees that are used for lumber production, or trees and shrubs that serve as view or wind screens, increased stature provides improved benefits in the forms of greater yield or improved screening. Crop species may also produce higher yields on larger cultivars, particularly those in which the vegetative portion of the plant is edible.

Plant size: large seedlings. Presently disclosed transcription factor genes, that produce large seedlings can be used to produce crops that become established faster. Large seedlings are generally hardier, less vulnerable to stress, and better able to out-compete weed species. Seedlings transformed with presently disclosed transcription factors, including G2346 and G2838, for example, have been shown to possess larger cotyledons and were more developmentally advanced than control plants. Rapid seedling development made possible by manipulating expression of these genes or their equivalogs is likely to reduce loss due to diseases particularly prevalent at the seedling stage (e.g., damping off) and is thus important for survivability of plants germinating in the field or in controlled environments.

Plant size: dwarfed plants. Presently disclosed transcription factor genes, including G24; G343, G353, G354, G362 , G370; G1008, G1277, G1543, G1794, G1958 and their equivalogs, for example, that can be used to decrease plant stature are likely to produce plants that are more resistant to damage by wind and rain, have improved lodging resistance, or more resistant to heat or low humidity or water deficit. Dwarf plants are also of significant interest to the ornamental horticulture industry, and particularly for home garden applications for which space availability may be limited.

Plant size: fruit size and number. Introduction of presently disclosed transcription factor genes that affect fruit size will have desirable impacts on fruit size and number, which may comprise increases in yield for fruit crops, or reduced fruit yield, such as when vegetative growth is preferred (e.g., with bushy ornamentals, or where fruit is undesirable, as with ornamental olive trees).

Leaf morphology: dark leaves. Color-affecting components in leaves include chlorophylls (generally green), anthocyanins (generally red to blue) and carotenoids (generally yellow to red). Transcription factor genes that increase these pigments in leaves, including G674, G912, G1063, G1357, G1452, G1482, G1499, G1792, G1863, G1888, G2143, G2557, G2838 and their equivalogs, may positively affect a plant's value to the ornamental horticulture industry. Variegated varieties, in particular, would show improved contrast. Other uses that result from overexpression of transcription factor genes include improvements in the nutritional value of foodstuffs. For example, lutein is an important nutraccutical; lutein-rich diets have been shown to help prevent age-related macular degeneration (ARMD), the leading cause of blindness in elderly people. Consumption of dark green leafy vegetables has been shown in clinical studies to reduce the risk of ARMD.

Enhanced chlorophyll and carotenoid levels could also improve yield in crop plants. Lutein, like other xanthophylls such as zeaxanthin and violaxanthin, is an essential component in the protection of the plant against the damaging effects of excessive light. Specifically, lutein contributes, directly or indirectly, to the rapid rise of non-photochemical quenching in plants exposed to high light. Crop plants engineered to contain higher levels of lutein could therefore have improved photo-protection, leading to less oxidative damage and better growth under high light (e.g., during long summer days, or at higher altitudes or lower latitudes than those at which a non-transformed plant would survive). Additionally, elevated chlorophyll levels increases photosynthetic capacity.

Leaf morphology: changes in leaf shape. Presently disclosed transcription factors produce marked and diverse effects on leaf development and shape. The transcription factors include G211, G353, G674, G736, G1063, G1146, G1357, G1452, G1494, G1543, G1863, G2143, G2144, and their equivalogs. At early stages of growth, transgenic seedlings have developed narrow, upward pointing leaves with long petioles, possibly indicating a disruption in circadian-clock controlled processes or nyctinastic movements. Other transcription factor genes can be used to alter leaf shape in a significant manner from wild type, some of which may find use in ornamental applications.

Leaf morphology: altered leaf size. Large leaves, such as those produced in plants overexpressing G189,G1451,G2430 and their functional equivalogs, generally increase plant biomass. This provides benefit for crops where the vegetative portion of the plant is the marketable portion.

Leaf morphology: light green and variegated leaves. Transcription factor genes such as G635, G1494, G2144 and their equivalogs that provide an altered appearance may positively affect a plant's value to the ornamental horticulture industry.

Leaf morphology: glossy leaves. Transcription factor genes such as G30, G1792, G2583 and their equivalogs that induce the formation of glossy leaves generally do so by elevating levels of epidermal wax. Thus, the genes could be used to engineer changes in the composition and amount of leaf surface components, including waxes. The ability to manipulate wax composition, amount, or distribution could modify plant tolerance to drought and low humidity, or resistance to insects or pathogens. Additionally, wax may be a valuable commodity in some species, and altering its accumulation and/or composition could enhance yield.

Seed morphology: altered seed coloration. Presently disclosed transcription factor genes, including G156, G2105, G2085 have also been used to modify seed color, which, along with the equivalogs of these genes, could provide added appeal to seeds or seed products.

Seed morphology: altered seed size and shape. The introduction of presently disclosed transcription factor genes into plants that increase (e.g., G450; G584; G1255; G2085; G2105; G2114) or decrease (e.g., G1040) the size of seeds may have a significant impact on yield and appearance, particularly when the product is the seed itself (e.g., in the case of grains, legumes, nuts, etc.). Seed size, in addition to seed coat integrity, thickness and permeability, seed water content and a number of other components including antioxidants and oligosaccharides, also affects affect seed longevity in storage, with larger seeds often being more desirable for prolonged storage.

Transcription factor genes that alter seed shape, including G1040, G1062, G1255 and their equivalogs may have both ornamental applications and improve or broaden the appeal of seed products.

Leaf biochemistry: increased leaf wax. Overexpression of transcription factors genes, including G975, G1792 and G2085 and their equivalogs, which results in increased leaf wax could be used to manipulate wax composition, amount, or distribution. These transcription factors can improve yield in those plants and crops from which wax is a valuable product. The genes may also be used to modify plant tolerance to drought and/or low humidity or resistance to insects, as well as plant appearance (glossy leaves). The effect of increased wax deposition on leaves of a plant like may improve water use efficiency. Manipulation of these genes may reduce the wax coating on sunflower seeds; this wax fouls the oil extraction system during sunflower seed processing for oil. For the latter purpose or any other where wax reduction is valuable, antisense or cosuppression of the transcription factor genes in a tissue-specific manner would be valuable.

Leaf biochemistry: leaf prenyl lipids, including tocopherol. Prenyl lipids play a role in anchoring proteins in membranes or membranous organelles. Thus modifying the prenyl lipid content of seeds and leaves could affect membrane integrity and function. One important group of prenyl lipids, the tocopherols, have both anti-oxidant and vitamin E activity. A number of presently disclosed transcription factor genes, including G214, G652, G748, G987, G1543, and G2509, have been shown to modify the tocopherol composition of leaves in plants, and these genes and their equivalogs may thus be used to alter prenyl lipid content of leaves.

Leaf biochemistry: increased leaf insoluble sugars. Overexpression of a number of presently disclosed transcription factors, including G211, resulted in plants with altered leaf insoluble sugar content. This transcription factor and its equivalogs that alter plant cell wall composition have several potential applications including altering food digestibility, plant tensile strength, wood quality, pathogen resistance and in pulp production. In particular, hemicellulose is not desirable in paper pulps because of its lack of strength compared with cellulose. Thus modulating the amounts of cellulose vs. hemicellulose in the plant cell wall is desirable for the paper/lumber industry. Increasing the insoluble carbohydrate content in various fruits, vegetables, and other edible consumer products will result in enhanced fiber content. Increased fiber content would not only provide health benefits in food products, but might also increase digestibility of forage crops. In addition, the hemicellulose and pectin content of fruits and berries affects the quality of jam and catsup made from them. Changes in hemicellulose and pectin content could result in a superior consumer product.

Leaf biochemistry: increased leaf anthoc anin. Several presently disclosed transcription factor genes may be used to alter anthocyanin production in numerous plant species. Expression of presently disclosed transcription factor genes that increase flavonoid production in plants, including anthocyanins and condensed tannins, may be used to alter in pigment production for horticultural purposes, and possibly increasing stress resistance. G362 , G663, G1482 and G1888 or their equivalogs, for example, could be used to alter anthocyanin production or accumulation. A number of flavonoids have been shown to have antimicrobial activity and could be used to engineer pathogen resistance. Several flavonoid compounds have health promoting effects such as inhibition of tumor growth, prevention of bone loss and prevention of the oxidation of lipids. Increased levels of condensed tannins, in forage legumes would be an important agronomic trait because they prevent pasture bloat by collapsing protein foams within the rumen. For a review on the utilities of flavonoids and their derivatives, refer to Dixon et al. (1999) Trends Plant Sci. 4: 394-400.

Leaf and seed biochemistry: altered fatty acid content. A number of the presently disclosed transcription factor genes have been shown to alter the fatty acid composition in plants, and seeds and leaves in particular. This modification suggests several utilities, including improving the nutritional value of seeds or whole plants. Dietary fatty acids ratios have been shown to have an effect on, for example, bone integrity and remodeling (see, for example, Weiler (2000) Pediatr. Res. 47:5692-697). The ratio of dietary fatty acids may alter the precursor pools of long-chain polyunsaturated fatty acids that serve as precursors for prostaglandin synthesis. In mammalian connective tissue, prostaglandins serve as important signals regulating the balance between resorption and formation in bone and cartilage. Thus dietary fatty acid ratios altered in seeds may affect the etiology and outcome of bone loss.

Transcription factors that reduce leaf fatty acids, for example, 16:3 fatty acids, may be used to control thylakoid membrane development, including proplastid to chloroplast development. The genes that encode these transcription factors might thus be useful for controlling the transition from proplastid to chromoplast in fruits and vegetables. It may also be desirable to change the expression of these genes to prevent cotyledon greening in Brassica napus or B. campestris to avoid green oil due to early frost.

A number of transcription factor genes are involved in mediating an aspect of the regulatory response to temperature. These genes may be used to alter the expression of desaturases that lead to production of 18:3 and 16:3 fatty acids, the balance of which affects membrane fluidity and mitigates damage to cell membranes and photosynthetic structures at high and low temperatures.

Seed biochemistry: modified seed oil and fatty acid content. The composition of seeds, particularly with respect to seed oil amounts and/or composition, is very important for the nutritional and caloric value and production of various food and feed products. Several of the presently disclosed transcription factor genes in seed lipid saturation that alter seed oil content could be used to improve the heat stability of oils or to improve the nutritional quality of seed oil, by, for example, reducing the number of calories in seed by decreasing oil or fatty acid content (e.g., G180; G192; G241; G1229; G1323; G1543), increasing the number of calories in animal feeds by increasing oil or fatty acid content (e.g. G162; G291; G427; G590; G598; G629, G715; G849; G1198, G1471; G1526; G1640; G1646, G1750; G1777; G1793; G1838; G1902; G1946; G1948; G2123; G2138; G2830), altering seed oil content (G504; G509; G519; G561; G567; G892; G961; G974; G1143; G1226; G1451; G1478; G1496; G1672; G1677; G1765; G2509; G2343), or altering the ratio of saturated to unsaturated lipids comprising the oils (e.g. G869; G1417; G2192).

Seed biochemistry: modified seed protein content. As with seed oils, the composition of seeds, particularly with respect to protein amounts and/or composition, is very important for the nutritional value and production of various food and feed products. A number of the presently disclosed transcription factor genes modify the protein concentrations in seeds, including G162; G226; G1323; G1419; G1818, which increase seed protein, G427; G1777; G1903; G1946, which decrease seed protein, and G162; G241; G509; G567; G597; G849; G892; G988; G1478; G1634; G1637; G1652; G1677; G1820; G1958; G2509; G2117; G2509, which alter seed protein content, would provide nutritional benefits, and may be used to prolong storage, increase seed pest or disease resistance, or modify germination rates.

Seed biochemistry: seed prenyl lipids. Prenyl lipids play a role in anchoring proteins in membranes or membranous organelles. Thus, modifying the prenyl lipid content of seeds and leaves could affect membrane integrity and function. A number of presently disclosed transcription factor genes have been shown to modify the tocopherol composition of plants. α-Tocopherol is better known as vitamin E. Tocopherols such as α- and γ-tocopherol both have anti-oxidant activity.

Seed biochemistry: seed glucosinolates. A number of glucosinolates have been shown to have anti-cancer activity; thus, increasing the levels or composition of these compounds by introducing several of the presently disclosed transcription factors, including G484 and G2340, can have a beneficial effect on human diet.

Glucosinolates are undesirable components of the oilseeds used in animal feed since they produce toxic effects. Low-glucosinolate varieties of canola, for example, have been developed to combat this problem. Glucosinolates form part of a plant's natural defense against insects. Modification of glucosinolate composition or quantity by introducing transcription factors that affect these characteristics can therefore afford increased protection from herbivores. Furthermore, in edible crops, tissue specific promoters can be used to ensure that these compounds accumulate specifically in tissues, such as the epidermis, which are not taken for consumption.

Seed biochemistry: increased seed anthocyanin. Several presently disclosed transcription factor genes may be used to alter anthocyanin production in the seeds of plants. As with leaf anthocyanins, expression of presently disclosed transcription factor genes that increase flavonoid (anthocyanins and condensed tannins) production in seeds, including G663 and its equivalogs, may be used to alter in pigment production for horticultural purposes, and possibly increasing stress resistance, antimicrobial activity and health promoting effects such as inhibition of tumor growth, prevention of bone loss and prevention of the oxidation of lipids.

Leaf and seed biochemistry: production of seed and leaf phytosterols: Presently disclosed transcription factor genes that modify levels of phytosterols in plants may have at least two utilities. First, phytosterols are an important source of precursors for the manufacture of human steroid hormones. Thus, regulation of transcription factor expression or activity could lead to elevated levels of important human steroid precursors for steroid semi-synthesis. For example, transcription factors that cause elevated levels of campesterol in leaves, or sitosterols and stigmasterols in seed crops, would be useful for this purpose. Phytosterols and their hydrogenated derivatives phytostanols also have proven cholesterol-lowering properties, and transcription factor genes that modify the expression of these compounds in plants would thus provide health benefits.

Root biochemistry: increased root anthocyanin. Presently disclosed transcription factor genes, including G663, may be used to alter anthocyanin production in the root of plants. As described above for seed anthocyanins, expression of presently disclosed transcription factor genes that increase flavonoid (anthocyanins and condensed tannins) production in seeds, including G663 and its equivalogs, may be used to alter in pigment production for horticultural purposes, and possibly increasing stress resistance, antimicrobial activity and health promoting effects such as inhibition of tumor growth, prevention of bone loss and prevention of the oxidation of lipids.

Light response/shade avoidance: altered cotyledon, hypocotyl, petiole development, altered leaf orientation, constitutive photomorphogenesis, photomorphogenesis in low light. Presently disclosed transcription factor genes, including G183; G354; G1322; G1331; G1488; G1494; G1794; G2144; and G2555, that modify a plant's response to light may be useful for modifying plant growth or development, for example, photomorphogenesis in poor light, or accelerating flowering time in response to various light intensities, quality or duration to which a non-transformed plant would not similarly respond. Examples of such responses that have been demonstrated include leaf number and arrangement, and early flower bud appearances. Elimination of shading responses may lead to increased planting densities with subsequent yield enhancement. As these genes may also alter plant architecture, they may find use in the ornamental horticulture industry.

Pigment: increased anthocyanin level in various plant organs and tissues. In addition to seed, leaves and roots, as mentioned above, several presently disclosed transcription factor genes can be used to alter anthocyanin levels in one or more tissues. The potential utilities of these genes include alterations in pigment production for horticultural purposes, and possibly increasing stress resistance, antimicrobial activity and health promoting effects such as inhibition of tumor growth, prevention of bone loss and prevention of the oxidation of lipids.

Miscellaneous biochemistry: diterpenes in leaves and other plant parts. Depending on the plant species, varying amounts of diverse secondary biochemicals (often lipophilic terpenes) are produced and exuded or volatilized by trichomes. These exotic secondary biochemicals, which are relatively easy to extract because they are on the surface of the leaf, have been widely used in such products as flavors and aromas, drugs, pesticides and cosmetics. Thus, the overexpression of genes that are used to produce diterpenes in plants may be accomplished by introducing transcription factor genes that induce said overexpression. One class of secondary metabolites, the diterpenes, can effect several biological systems such as tumor progression, prostaglandin synthesis and tissue inflammation. In addition, diterpenes can act as insect pheromones, termite allomones, and can exhibit neurotoxic, cytotoxic and antimitotic activities. As a result of this functional diversity, diterpenes have been the target of research several pharmaceutical ventures. In most cases where the metabolic pathways are impossible to engineer, increasing trichome density or size on leaves may be the only way to increase plant productivity.

Miscellaneous biochemistry: production of miscellaneous secondary metabolites. Microarray data suggests that flux through the aromatic amino acid biosynthetic pathways and primary and secondary metabolite biosynthetic pathways are up-regulated. Presently disclosed transcription factors have been shown to be involved in regulating alkaloid biosynthesis, in part by up-regulating the enzymes indole-3-glycerol phosphatase and strictosidine synthase. Phenylalanine ammonia lyase, chalcone synthase and trans-cinnamate mono-oxygenase are also induced, and are involved in phenylpropenoid biosynthesis.
Antisense and Co-suppression

In addition to expression of the nucleic acids of the invention as gene replacement or plant phenotype modification nucleic acids, the nucleic acids are also useful for sense and anti-sense suppression of expression, e.g., to down-regulate expression of a nucleic acid of the invention, e.g., as a further mechanism for modulating plant phenotype. That is, the nucleic acids of the invention, or subsequences or anti-sense sequences thereof, can be used to block expression of naturally occurring homologous nucleic acids. A variety of sense and anti-sense technologies are known in the art, e.g., as set forth in Lichtenstein and Nellen (1997) Antisense Technology: A Practical Approach IRL Press at Oxford University Press, Oxford, U.K. Antisense regulation is also described in Crowley et al. (1985) Cell 43: 633-641; Rosenberg et al. (1985) Nature 313: 703-706; Preiss et al. (1985) Nature 313: 27-32; Melton (1985) Proc. Natl. Acad. Sci. 82: 144-148; Izant and Weintraub (1985) Science 229: 345-352; and Kim and Wold (1985) Cell 42: 129-138. Additional methods for antisense regulation are known in the art. Antisense regulation has been used to reduce or inhibit expression of plant genes in, for example in European Patent Publication No. 271988. Antisense RNA may be used to reduce gene expression to produce a visible or biochemical phenotypic change in a plant (Smith et al. (1988) Nature, 334: 724-726; Smith et al. (1990) Plant Mol. Biol. 14: 369-379). In general, sense or anti-sense sequences are introduced into a cell, where they are optionally amplified, e.g., by transcription. Such sequences include both simple oligonucleotide sequences and catalytic sequences such as ribozymes.

For example, a reduction or elimination of expression (i.e., a “knock-out”) of a transcription factor or transcription factor homolog polypeptide in a transgenic plant, e.g., to modify a plant trait, can be obtained by introducing an antisense construct corresponding to the polypeptide of interest as a cDNA. For antisense suppression, the transcription factor or homolog cDNA is arranged in reverse orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector. The introduced sequence need not be the full length cDNA or gene, and need not be identical to the cDNA or gene found in the plant type to be transformed. Typically, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous transcription factor sequence will be needed for effective antisense suppression. While antisense sequences of various lengths can be utilized, preferably, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. Preferably, the length of the antisense sequence in the vector will be greater than 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous transcription factor gene in the plant cell.

Suppression of endogenous transcription factor gene expression can also be achieved using a ribozyme. Ribozymes are RNA molecules that possess highly specific endoribonuclease activity. The production and use of ribozymes are disclosed in U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,543,508. Synthetic ribozyme sequences including antisense RNAs can be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that hybridize to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.

Vectors in which RNA encoded by a transcription factor or transcription factor homolog cDNA is over-expressed can also be used to obtain co-suppression of a corresponding endogenous gene, e.g., in the manner described in U.S. Pat. No. 5,231,020 to Jorgensen. Such co-suppression (also termed sense suppression) does not require that the entire transcription factor cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous transcription factor gene of interest. However, as with antisense suppression, the suppressive efficiency will be enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous transcription factor gene is increased.

Vectors expressing an untranslatable form of the transcription factor mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation) can also be used to suppress expression of an endogenous transcription factor, thereby reducing or eliminating its activity and modifying one or more traits. Methods for producing such constructs are described in U.S. Pat. No. 5,583,021. Preferably, such constructs are made by introducing a premature stop codon into the transcription factor gene. Alternatively, a plant trait can be modified by gene silencing using double-strand RNA (Sharp (1999) Genes and Development 13: 139-141). Another method for abolishing the expression of a gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a transcription factor or transcription factor homolog gene. Plants containing a single transgene insertion event at the desired gene can be crossed to generate homozygous plants for the mutation. Such methods are well known to those of skill in the art (See for example Koncz et al. (1992) Methods in Arabidopsis Research, World Scientific Publishing Co. Pte. Ltd., River Edge, N.J.).

Alternatively, a plant phenotype can be altered by eliminating an endogenous gene, such as a transcription factor or transcription factor homolog, e.g., by homologous recombination (Kempin et al. (1997) Nature 389: 802-803).

A plant trait can also be modified by using the Cre-lox system (for example, as described in U.S. Pat. No. 5,658,772). A plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.

The polynucleotides and polypeptides of this invention can also be expressed in a plant in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means, such as, for example, by ectopically expressing a gene by T-DNA activation tagging (Ichikawa et al. (1997) Nature 390 698-701; Kakimoto et al. (1996) Science 274: 982-985). This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated. In another example, the transcriptional machinery in a plant can be modified so as to increase transcription levels of a polynucleotide of the invention (See, e.g., PCT Publications WO 96/06166 and WO 98/53057 which describe the modification of the DNA-binding specificity of zinc finger proteins by changing particular amino acids in the DNA-binding motif).

The transgenic plant can also include the machinery necessary for expressing or altering the activity of a polypeptide encoded by an endogenous gene, for example, by altering the phosphorylation state of the polypeptide to maintain it in an activated state.

Transgenic plants (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides of the invention and/or expressing the polypeptides of the invention can be produced by a variety of well established techniques as described above. Following construction of a vector, most typically an expression cassette, including a polynucleotide, e.g., encoding a transcription factor or transcription factor homolog, of the invention, standard techniques can be used to introduce the polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. Optionally, the plant cell, explant or tissue can be regenerated to produce a transgenic plant.

The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al., Eds., (1984) Handbook of Plant Cell Culture—Crop Species, Macmillan Publ. Co., New York, N.Y.; Shimamoto et al. (1989) Nature 338: 274-276; Fromm et al. (1990) Bio/Technol. 8: 833-839; and Vasil et al. (1990) Bio/Technol. 8: 429-434.

Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens mediated transformation. Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence.

Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and which are herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.

After transformed plants are selected and grown to maturity, those plants showing a modified trait are identified. The modified trait can be any of those traits described above. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.

Integrated Systems—Sequence Identity

Additionally, the present invention may be an integrated system, computer or computer readable medium that comprises an instruction set for determining the identity of one or more sequences in a database. In addition, the instruction set can be used to generate or identify sequences that meet any specified criteria. Furthermore, the instruction set may be used to associate or link certain functional benefits, such improved characteristics, with one or more identified sequence.

For example, the instruction set can include, e.g., a sequence comparison or other alignment program, e.g., an available program such as, for example, the Wisconsin Package Version 10.0, such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, Wis.). Public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PHYTOSEQ sequence database (Incyte Genomics, Palo Alto, Calif.) can be searched.

Alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482-489, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448, by computerized implementations of these algorithms. After alignment, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window can be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions. A description of the method is provided in Ausubel et al. supra.

A variety of methods for determining sequence relationships can be used, including manual alignment and computer assisted sequence alignment and analysis. This later approach is a preferred approach in the present invention, due to the increased throughput afforded by computer assisted methods. As noted above, a variety of computer programs for performing sequence alignment are available, or can be produced by one of skill.

One example algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available, e.g., through the National Library of Medicine's National Center for Biotechnology Information (ncbi.nlm.nih; see at world wide web (www) National Institutes of Health US government (gov) website). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. 89: 10915-10919). Unless otherwise indicated, “sequence identity” here refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter “off” (see, for example, NIH NLM NCBI website at ncbi.nlm.nih, supra).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g. Karlin and Altschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence (and, therefore, in this context, homologous) if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, or less than about 0.01, and or even less than about 0.001. An additional example of a useful sequence alignment algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. The program can align, e.g., up to 300 sequences of a maximum length of 5,000 letters.

The integrated system, or computer typically includes a user input interface allowing a user to selectively view one or more sequence records corresponding to the one or more character strings, as well as an instruction set which aligns the one or more character strings with each other or with an additional character string to identify one or more region of sequence similarity. The system may include a link of one or more character strings with a particular phenotype or gene function. Typically, the system includes a user readable output element that displays an alignment produced by the alignment instruction set.

The methods of this invention can be implemented in a localized or distributed computing environment. In a distributed environment, the methods may implemented on a single computer comprising multiple processors or on a multiplicity of computers. The computers can be linked, e.g. through a common bus, but more preferably the computer(s) are nodes on a network. The network can be a generalized or a dedicated local or wide-area network and, in certain preferred embodiments, the computers may be components of an intra-net or an internet.

Thus, the invention provides methods for identifying a sequence similar or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given plant phenotype or gene function with a sequence. In the methods, a sequence database is provided (locally or across an inter or intra net) and a query is made against the sequence database using the relevant sequences herein and associated plant phenotypes or gene functions.

Any sequence herein can be entered into the database, before or after querying the database. This provides for both expansion of the database and, if done before the querying step, for insertion of control sequences into the database. The control sequences can be detected by the query to ensure the general integrity of both the database and the query. As noted, the query can be performed using a web browser based interface. For example, the database can be a centralized public database such as those noted herein, and the querying can be done from a remote terminal or computer across an internet or intranet.

Any sequence herein can be used to identify a similar, homologous, paralogous, or orthologous sequence in another plant. This provides means for identifying endogenous sequences in other plants that may be useful to alter a trait of progeny plants, which results from crossing two plants of different strain. For example, sequences that encode an ortholog of any of the sequences herein that naturally occur in a plant with a desired trait can be identified using the sequences disclosed herein. The plant is then crossed with a second plant of the same species but which does not have the desired trait to produce progeny which can then be used in further crossing experiments to produce the desired trait in the second plant. Therefore the resulting progeny plant contains no transgenes; expression of the endogenous sequence may also be regulated by treatment with a particular chemical or other means, such as EMR. Some examples of such compounds well known in the art include: ethylene; cytokinins; phenolic compounds, which stimulate the transcription of the genes needed for infection; specific monosaccharides and acidic environments which potentiate vir gene induction; acidic polysaccharides which induce one or more chromosomal genes; and opines; other mechanisms include light or dark treatment (for a review of examples of such treatments, see, Winans (1992) Microbiol. Rev. 56: 12-31; Eyal et al. (1992) Plant Mol. Biol. 19: 589-599; Chrispeels et al. (2000) Plant Mol. Biol. 42: 279-290; Piazza et al. (2002) Plant Physiol. 128: 1077-1086).

Table 7 lists sequences discovered to be orthologous to a number of representative transcription factors of the present invention. The column headings include the transcription factors listed by SEQ ID NO; corresponding Gene ID (GID) numbers; the species from which the orthologs to the transcription factors are derived; the type of sequence (i.e., DNA or protein) discovered to be orthologous to the transcription factors; and the SEQ ID NO of the orthologs, the latter corresponding to the ortholog SEQ ID NOs listed in the Sequence Listing.

TABLE 7
Orthologs of Representative Arabidopsis Transcription Factor Genes
SEQ ID No. of
Nucleotide
SEQ ID NO: GID NO of Encoding
of Ortholog Sequence type Orthologous Orthologous
or Nucleotide used for Arabidopsis Arabidopsis
Encoding Ortholog Species from Which determination Transcription Transcription
Ortholog GID NO Ortholog is Derived (DNA or Protein) Factor Factor
459 Glycine max DNA G8 1
460 Glycine max DNA G8 1
461 Glycine max DNA G8 1
462 Glycine max DNA G8 1
463 Oryza sativa DNA G8 1
464 Zea mays DNA G8 1
465 Zea mays DNA G8 1
466 Zea mays DNA G8 1
467 Oryza sativa PRT G8 1
468 Glycine max DNA G19 3
469 Glycine max DNA G19 3
470 Glycine max DNA G19 3
471 Glycine max DNA G19 3
472 Oryza sativa DNA G19 3
473 Oryza sativa DNA G19 3
474 Oryza sativa DNA G19 3
475 Zea mays DNA G19 3
476 Zea mays DNA G19 3
477 Glycine max DNA G22 5
478 Glycine max DNA G22 5
479 Glycine max DNA G24 7
480 Glycine max DNA G24 7
481 Glycine max DNA G24 7
482 Glycine max DNA G24 7
483 Glycine max DNA G24 7
484 Glycine max DNA G24 7
485 Glycine max DNA G24 7
486 Oryza sativa DNA G24 7
487 Zea mays DNA G24 7
488 Oryza sativa PRT G24 7
489 Oryza sativa PRT G24 7
490 Oryza sativa PRT G24 7
491 Glycine max DNA G28 9
492 Glycine max DNA G28 9
493 Glycine max DNA G28 9
494 Glycine max DNA G28 9
495 Glycine max DNA G28 9
496 Glycine max DNA G28 9
497 Glycine max DNA G28 9
498 Glycine max DNA G28 9
499 Oryza sativa DNA G28 9
500 Zea mays DNA G28 9
501 Oryza sativa PRT G28 9
502 Oryza sativa PRT G28 9
503 Mesembryanthemum PRT G28 9
crystallinum
504 Glycine max DNA G47, G2133 11, 407
505 Oryza sativa PRT G47, G2133 11, 407
506 Glycine max DNA G157, G859, 15, 165, 349,
G1842, G1843 351
507 Glycinemax DNA G175, G877 19, 173
508 Oryza sativa DNA G175, G877 19, 173
509 Zea mays DNA G175, G877 19, 173
510 Zeamays DNA G175, G877 19, 173
511 Zea mays DNA G175, G877 19, 173
512 Oryza sativa PRT G175, G877 19, 173
513 Oryza sativa PRT G175, G877 19, 173
514 Oryza sativa PRT G175, G877 19, 173
515 Nicotiana tabacum PRT G175, G877 19, 173
516 Glycine max DNA G180 21
517 Glycine max DNA G180 21
518 Oryza sativa DNA G180 21
519 Zea mays DNA G180 21
520 Solanum tuberosum DNA G180 21
521 Oryza sativa PRT G180 21
522 Capsella rubella PRT G183 23
523 Glycine max DNA G188 25
524 Zea mays DNA G188 25
525 Oryza sativa PRT G188 25
526 Oryza sativa PRT G188 25
527 Glycine max DNA G189 27
528 Nicotiana tabacum PRT G189 27
529 Glycine max DNA G192 29
530 Oryza sativa PRT G192 29
531 Glycine max DNA G196 31
532 Zea mays DNA G196 31
533 Zea mays DNA G196 31
534 Oryza sativa PRT G196 31
535 Oryza sativa PRT G196 31
536 Oryza sativa PRT G196 31
537 Oryza sativa PRT G196 31
538 Glycine max DNA G211 33
539 Oryza sativa DNA G211 33
540 Oryza sativa PRT G211 33
541 Glycine max DNA G214, G680 35, 145
542 Glycine max DNA G214, G680 35, 145
543 Glycine max DNA G214, G680 35, 145
544 Glycine max DNA G214, G680 35, 145
545 Oryza sativa DNA G214, G680 35, 145
546 Oryza sativa DNA G214, G680 35, 145
547 Zea mays DNA G214, G680 35, 145
548 Zea mays DNA G214, G680 35, 145
549 Zea mays DNA G214, G680 35, 145
550 Glycine max DNA G226, G682 37, 147
551 Glycine max DNA G226 37
552 Glycine max DNA G226, G682 37, 147
553 Glycine max DNA G226, G682 37, 147
554 Glycine max DNA G226, G682 37, 147
555 Oryza sativa DNA G226, G682 37, 147
556 Zea mays DNA G226, G682 37, 147
557 Zea mays DNA G226, G682 37, 147
558 Oryza sativa PRT G226, G682 37, 147
559 Oryza sativa PRT G226, G682 37, 147
560 Glycine max DNA G241 39
561 Glycine max DNA G241 39
562 Glycine max DNA G241 39
563 Oryza sativa DNA G241 39
564 Zea mays DNA G241 39
565 Zea mays DNA G241 39
566 Zea mays DNA G241 39
567 Zea mays DNA G241 39
568 Zea mays DNA G241 39
569 Nicotiana tabacum PRT G241 39
570 Glycine max DNA G254 43
571 Glycine max DNA G256 45
572 Glycine max DNA G256 45
573 Glycine max DNA G256 45
574 Glycine max DNA G256 45
575 Glycine max DNA G256 45
576 Glycine max DNA G256 45
577 Glycine max DNA G256 45
578 Oryza sativa DNA G256 45
579 Zea mays DNA G256 45
580 Zea mays DNA G256 45
581 Zea mays DNA G256 45
582 Zea mays DNA G256 45
583 Zea mays DNA G256 45
584 Zea mays DNA G256 45
585 G3500 Lycopersicon DNA G256 45
esculentum
586 G3501 Lycopersicon DNA G256 45
esculentum
587 G3385 Oryza sativa PRT G256 45
588 G3386 Oryza sativa PRT G256 45
589 Oryza sativa PRT G256 45
590 G3384 Oryza sativa PRT G256 45
591 Oryza sativa PRT G256 45
592 G3502 Oryza sativa japonica PRT G256 45
593 G3500 Lycopersicon PRT G256 45
esculentum
594 G3501 Lycopersicon PRT G256 45
esculentum
595 Oryza sativa DNA G278 47
596 Zea mays DNA G278 47
597 Oryza sativa PRT G278 47
598 Glycine max DNA G312 53
599 Zea mays DNA G312 53
600 Euphorbia esula DNA G312 53
601 Glycine max DNA G325 55
602 Glycine max DNA G343 57
603 Glycine max DNA G343 57
604 Glycine max DNA G343 57
605 Oryza sativa DNA G343 57
606 Oryza sativa DNA G343 57
607 Oryza sativa PRT G343 57
608 Oryza sativa PRT G343 57
609 Oryza sativa PRT G343 57
610 Glycine max DNA G353, G354 59, 61
611 Glycine max DNA G353, G354 59, 61
612 Glycine max DNA G353, G354 59, 61
613 Oryza sativa DNA G353, G354 59, 61
614 Zea mays DNA G353, G354 59, 61
615 Zea mays DNA G353, G354 59, 61
616 Zea mays DNA G353, G354 59, 61
617 Zea mays DNA G353, G354 59, 61
618 Zea mays DNA G353, G354 59, 61
619 Zea mays DNA G353, G354 59, 61
620 Zea mays DNA G353, G354 59, 61
621 Oryza sativa PRT G353, G354 59, 61
622 Oryza sativa PRT G353, G354 59, 61
623 Oryza sativa PRT G353, G354 59, 61
624 Oryza sativa PRT G353, G354 59, 61
625 Oryza sativa PRT G353, G354 59, 61
626 Oryza sativa PRT G353, G354 59, 61
627 Glycine max DNA G361, G362 63, 65
628 Glycine max DNA G361, G362 63, 65
629 Glycine max DNA G361 63
630 Glycine max DNA G361, G362 63, 65
631 Glycine max DNA G361, G362 63, 65
632 Oryza sativa DNA G361, G362 63, 65
633 Zea mays DNA G361, G362 63, 65
634 Zea mays DNA G361, G362 63, 65
635 Oryza sativa PRT G361, G362 63, 65
636 Oryza sativa PRT G361, G362 63,65
637 Oryza sativa PRT G361, G362 63, 65
638 Oryza sativa PRT G361, G362 63, 65
639 Oryza sativa PRT G361, G362 63, 65
640 Glycine max DNA G390, G391, 69, 71, 77
G438
641 Glycine max DNA G390, G391, 69, 71, 77
G438
642 Glycine max DNA G390, G391, 69, 71, 77
G438
643 Glycine max DNA G390, G391, 69, 71, 77
G438
644 Glycine max DNA G390, G391, 69, 71, 77
G438
645 Glycine max DNA G390, G391, 69, 71, 77
G438
646 Glycine max DNA G390, G391, 69, 71, 77
G438
647 Glycine max DNA G390, G391 69, 71
648 Glycine max DNA G390, G391, 69, 71, 77
G438
649 Glycine max DNA G390, G391, 69, 71, 77
G438
650 Oryza sativa DNA 6390 69
651 Oryza sativa DNA G390, G438 69, 77
652 Zea mays DNA G390, G391, 69, 71, 77
G438
653 Zeamays DNA G390, G391, 69, 71, 77
G438
654 Zea mays DNA G390, G391, 69, 71, 77
G438
655 Zea mays DNA G390, G391 69, 71
656 Zea mays DNA G390, G391, 69, 71, 77
G438
657 Zea mays DNA G390, G391, 69, 71, 77
G438
658 Zea mays DNA G390, G391, 69, 71, 77
G438
659 Zea mays DNA G390, G391, 69, 71, 77
G438
660 Zea mays DNA G390, G391, 69, 71, 77
G438
661 Zea mays DNA G390, G391, 69, 71, 77
G438
662 Zea mays DNA G390, G391, 69, 71, 77
G438
663 Lycopersicon DNA G390, G391, 69, 71, 77
esculentum G438
664 Oryza sativa DNA G391, G438 71, 77
665 Oryza sativa PRT G390, G391, 69, 71, 77
G438
666 Oryza sativa PRT G390, G391, 69, 71, 77
G438
667 Oryza sativa PRT G390, G391, 69, 71, 77
G438
668 Oryza sativa PRT G390, G391, 69, 71, 77
G438
669 Physcomitrella PRT G391 71
patens
670 Glycine max DNA G409 73
671 Glycine max DNA G409 73
672 Glycine max DNA G409 73
673 Glycine max DNA G409 73
674 Glycine max DNA G409 73
675 Glycine max DNA G409 73
676 Glycine max DNA G409 73
677 Glycine max DNA G409 73
678 Oryza sativa DNA G409 73
679 Oryza sativa DNA G409 73
680 Oryza sativa DNA G409 73
681 Zea mays DNA G409 73
682 Zea mays DNA G409 73
683 Zea mays DNA G409 73
684 Zea mays DNA G409 73
685 Zea mays DNA G409 73
686 Zea mays DNA G409 73
687 Zea mays DNA G409 73
688 Glycine max DNA G427 75
689 Glycine max DNA G427 75
690 Glycine max DNA G427 75
691 Glycine max DNA G427 75
692 Glycine max DNA G427 75
693 Glycine max DNA G427 75
694 Glycine max DNA G427 75
695 Glycine max DNA G427 75
696 Glycine max DNA G427 75
697 Glycine max DNA G427 75
698 Oryza sativa DNA G427 75
699 Zea mays DNA G427 75
700 Zea mays DNA G427 75
701 Zea mays DNA G427 75
702 Zea mays DNA G427 75
703 Zea mays DNA G427 75
704 Zea mays DNA G427 75
705 Zea mays DNA G427 75
706 Zea mays DNA G427 75
707 Zea mays DNA G427 75
708 Oryza sativa PRT G427 75
709 Oryza sativa PRT G427 75
710 Oryza sativa PRT G427 75
711 Malus × domestica PRT G427 75
712 Nicotiana tabacum PRT G427 75
713 Lycopersicon PRT G427 75
esculentum
714 Glycine max DNA G438 77
715 Oryza sativa DNA G438 77
716 Oryza sativa DNA G438 77
717 Oryza sativa DNA G438 77
718 Oryza sativa DNA G438 77
719 Zea mays DNA G438 77
720 Physcomitrella PRT G438 77
patens
721 Oryza sativa PRT G438 77
722 Glycine max DNA G450 79
723 Glycine max DNA G450 79
724 Glycine max DNA G450 79
725 Glycine max DNA G450 79
726 Glycine max DNA G450 79
727 Glycine max DNA G450 79
728 Glycine max DNA G450 79
729 Glycine max DNA G450 79
730 Glycine max DNA G450 79
731 Oryza sativa DNA G450 79
732 Oryza sativa DNA G450 79
733 Zea mays DNA G450 79
734 Zea mays DNA G450 79
735 Zea mays DNA G450 79
736 Oryza sativa PRT G450 79
737 Oryza sativa PRT G450 79
738 Oryza sativa PRT G450 79
739 Oryza sativa PRT G450 79
740 Oryza sativa DNA G464 81
741 Zea mays DNA G464 81
742 Oryza sativa PRT G464 81
743 Glycine max DNA G470 83
744 Oryza sativa DNA G470 83
745 Oryza sativa DNA G470 83
746 Glycine max DNA G481, G482 87, 89
747 Glycine max DNA G481, G482 87, 89
748 Glycine max DNA G481, G482 87, 89
749 Glycine max DNA G481, G482 87, 89
750 Glycine max DNA G481, G482 87, 89
751 Glycine max DNA G481, G482 87, 89
752 Glycine max DNA G481, G482 87, 89
753 Glycine max DNA G481, G482 87, 89
754 Glycine max DNA G481 87
755 Glycine max DNA G481 87
756 Oryza sativa DNA G481 87
757 Oryza sativa DNA G481, G482 87, 89
758 Zea mays DNA G481 87
759 Zea mays DNA G481, G482 87, 89
760 Zen mays DNA G481, G482 87, 89
761 Zea mays DNA G481, G482 87, 89
762 Zea mays DNA G481, G482 87, 89
763 Zea mays DNA G481, G482 87, 89
764 Zea mays DNA G481, G482 87, 89
765 Zea mays DNA G481, G482 87, 89
766 Zen mays DNA G481, G482 87, 89
767 Zea mays DNA G481, G482 87, 89
768 Gossypium arboreum DNA G481, G482 87, 89
769 Glycine max DNA G481, G482 87, 89
770 Gossypium hirsutum DNA G481, G482 87, 89
771 Lycopersicon DNA G481, G482 87, 89
esculentum
772 Lycopersicon DNA G481, G482 87, 89
esculentum
773 Medicago truncatula DNA G481, G482 87, 89
774 Lycopersicon DNA G481, G482 87, 89
esculentum
775 Solanum tuberosum DNA G481, G482 87, 89
776 Triticum aestivum DNA G481, G482 87, 89
777 Hordeum vulgare DNA G481, G482 87, 89
778 Triticum DNA G481, G482 87, 89
monococcum
779 Glycine max DNA G482 89
780 Oryza sativa PRT G481, G482 87, 89
781 Oryza sativa PRT G481, G482 87, 89
782 Oryza sativa PRT G481, G482 87, 89
783 Oryza sativa PRT G481, G482 87, 89
784 Oryza sativa PRT G481, G482 87, 89
785 Zea mays PRT G481, G482 87, 89
786 Zen mays PRT G481, G482 87, 89
787 Oryza sativa PRT G481, G482 87, 89
788 Oryza sativa PRT G481, G482 87, 89
789 Oryza sativa PRT G481, G482 87, 89
790 Oryza sativa PRT G481, G482 87, 89
791 Oryza sativa PRT G481, G482 87, 89
792 Oryza sativa PRT G481, G482 87, 89
793 Oryza sativa PRT G481, G482 87, 89
794 Oryza sativa PRT G481, G482 87, 89
795 Oryza sativa PRT G481, G482 87, 89
796 Oryza sativa PRT G481, G482 87, 89
797 Glycine max PRT G481, 6482 87, 89
798 Glycine max PRT G481, G482 87, 89
799 Glycine max PRT G481, G482 87, 89
800 Glycine max PRT G481, G482 87, 89
801 Glycine max PRT G481, G482 87, 89
802 Glycine max PRT G481, G482 87, 89
803 Glycine max PRT G481, G482 87, 89
804 Zea mays PRT G481, G482 87, 89
805 Zea mays PRT G481, G482 87, 89
806 Zea mays PRT G481, G482 87, 89
807 Zea mays PRT G481, G482 87, 89
808 Glycine max DNA G484 91
809 Glycine max DNA G484 91
810 Glycine max DNA G484 91
811 Glycine max DNA G484 91
812 Glycine max DNA G484 91
813 Glycine max DNA G484 91
814 Glycine max DNA G484 91
815 Glycine max DNA G484 91
816 Glycine max DNA G484 91
817 Glycine max DNA G484 91
818 Oryza sativa DNA G484 91
819 Zea mays DNA G484 91
820 Zea mays DNA G484 91
821 Zea mays DNA G484 91
822 Zea mays DNA G484 91
823 Zea mays DNA G484 91
824 Oryza sativa PRT G484 91
825 Glycine max DNA G489 93
826 Glycine max DNA G489 93
827 Glycine max DNA G489 93
828 Glycine max DNA G489 93
829 Glycine max DNA G489 93
830 Glycine max DNA G489 93
831 Glycine max DNA G489 93
832 Oryza sativa DNA G489 93
833 Oryza sativa DNA G489 93
834 Zea mays DNA G489 93
835 Oryza sativa PRT G489 93
836 Oryza sativa PRT G489 93
837 Oryza sativa PRT G489 93
838 Glycine max DNA G504 97
839 Glycine max DNA G504 97
840 Glycine max DNA G504 97
841 Glycine max DNA G504 97
842 Glycine max DNA G504 97
843 Glycine max DNA G504 97
844 Glycine max DNA G504 97
845 Oryza sativa DNA G504 97
846 Oryza sativa DNA G504 97
847 Zea mays DNA G504 97
848 Zea mays DNA G504 97
849 Zea mays DNA G504 97
850 Zea mays DNA G504 97
851 Oryza sativa PRT G504 97
852 Oryza sativa PRT G504 97
853 Oryza sativa PRT G504 97
854 Oryza sativa PRT G504 97
855 Lycopersicon DNA G509 99
esculentum
856 Glycine max DNA G509 99
857 Glycine max DNA G509 99
858 Glycine max DNA G509 99
859 Oryza sativa DNA G509 99
860 Oryza sativa DNA G509 99
861 Zea mays DNA G509 99
862 Zea mays DNA G509 99
863 Zea mays DNA G509 99
864 Zea mays DNA G509 99
865 Oryza sativa PRT G509 99
866 Oryza sativa PRT G509 99
867 Oryza sativa PRT G509 99
868 Glycine max DNA G519 101
869 Glycine max DNA G519 101
870 Glycine max DNA G519 101
871 Glycine max DNA G519 101
872 Glycine max DNA G519 101
873 Glycine max DNA G519 101
874 Glycine max DNA G519 101
875 Glycine max DNA G519 101
876 Glycine max DNA G519 101
877 Oryza sativa DNA G519 101
878 Oryza sativa DNA G519 101
879 Oryza sativa DNA G519 101
880 Zea mays DNA G519 101
881 Zea mays DNA G519 101
882 Zea mays DNA G519 101
883 Zea mays DNA G519 101
884 Zea mays DNA G519 101
885 Zea mays DNA G519 101
886 Zea mays DNA G519 101
887 Zea mays DNA G519 101
888 Zea mays DNA G519 101
889 Zea mays DNA G519 101
890 Oryza sativa PRT G519 101
891 Oryza sativa PRT G519 101
892 Glycine max DNA G545 103
893 Glycine max DNA G545 103
894 Glycine max DNA G545 103
895 Glycine max DNA G545 103
896 Glycine max DNA G545 103
897 Glycine max DNA G545 103
898 Glycine max DNA G545 103
899 Oryza sativa DNA G545 103
900 Zea mays DNA G545 103
901 Zea mays DNA G545 103
902 Zea mays DNA G545 103
903 Oryza sativa PRT G545 103
904 Oryza sativa PRT G545 103
905 Oryza sativa PRT G545 103
906 Oryza sativa PRT G545 103
907 Datisca glomerata PRT G545 103
908 Oryza sativa DNA G546 105
909 Zea mays DNA G561 107
910 Sinapis alba PRT G561 107
911 Raphanus sativus PRT G561 107
912 Brassica napus PRT G561 107
913 Brassica napus PRT G561 107
914 Glycine max DNA G562 109
915 Glycine max DNA G562 109
916 Glycine max DNA G562 109
917 Glycine max DNA G562 109
918 Glycine max DNA G562 109
919 Zea mays DNA G562 109
920 Zea mays DNA G562 109
921 Zea mays DNA G562 109
922 Oryza sativa PRT G562 109
923 Oryza sativa PRT G562 109
924 Glycine max DNA G567 111
925 Oryza sativa DNA G567 111
926 Oryza sativa PRT G567 111
927 Glycine max DNA G568 113
928 Glycine max DNA G568 113
929 Oryza sativa DNA G568 113
930 Oryza sativa DNA G568 113
931 Oryza sativa DNA G568 113
932 Zea mays DNA G568 113
933 Oryza sativa PRT G568 113
934 Populus balsamifera PRT G568 113
subsp. trichocarpa ×
Populus deltoides
935 Glycine max DNA G584 115
936 Glycine max DNA G584 115
937 Glycine max DNA G584 115
938 Glycine max DNA G584 115
939 Glycine max DNA G584 115
940 Zea mays DNA G584 115
941 Zea mays DNA G584 115
942 Zea mays DNA G584 115
943 Oryza sativa PRT G584 115
944 Glycine max DNA G585 117
945 Glycine max DNA G585 117
946 Glycine max DNA G585 117
947 Glycine max DNA G585 117
948 Oryza sativa DNA G585 117
949 Zea mays DNA G585 117
950 Zea mays DNA G585 117
951 Zea mays DNA G585 117
952 Zea mays DNA G585 117
953 Oryza sativa PRT G585 117
954 Oryza sativa PRT G585 117
955 Oryza sativa PRT G585 117
956 Oryza sativa PRT G585 117
957 Oryza sativa PRT G585 117
958 Oryza sativa PRT G585 117
959 Gossypium hirsutum PRT G585 117
960 Antirrhinum majus PRT G585 117
961 Glycine max DNA G590 119
962 Glycine max DNA G590 119
963 Glycine max DNA G590 119
964 Oryza sativa DNA G590 119
965 Zea mays DNA G590 119
966 Oryza sativa PRT G590 119
967 Oryza sativa PRT G590 119
968 Oryza sativa DNA G597 123
969 Oryza sativa DNA G597 123
970 Oryza sativa DNA G597 123
971 Zea mays DNA G597 123
972 Zea mays DNA G597 123
973 Zea mays DNA G597 123
974 Zea mays DNA G597 123
975 Zea mays DNA G597 123
976 Zea mays DNA G597 123
977 Zea mays DNA G597 123
978 Zea mays DNA G597 123
979 Zea mays DNA G597 123
980 Zea mays DNA G597 123
981 Oryza sativa DNA G634 127
982 Oryza sativa DNA G634 127
983 Oryza sativa DNA G634 127
984 Zea mays DNA G634 127
985 Zea mays DNA G634 127
986 Zea mays DNA G634 127
987 Oryza sativa PRT G634 127
988 Oryza sativa PRT G634 127
989 Glycine max DNA G635 129
990 Glycine max DNA G635 129
991 Oryza sativa DNA G635 129
992 Oryza sativa DNA G635 129
993 Zea mays DNA G635 129
994 Oryza sativa PRT G635 129
995 Glycine max DNA G636 131
996 Glycine max DNA G636 131
997 Glycine max DNA G636 131
998 Glycine max DNA G636 131
999 Glycine max DNA G636 131
1000 Glycine max DNA G636 131
1001 Glycine max DNA G636 131
1002 Glycine max DNA G636 131
1003 Oryza sativa DNA G636 131
1004 Oryza sativa DNA G636 131
1005 Oryza sativa DNA G636 131
1006 Oryza sativa DNA G636 131
1007 Zea mays DNA G636 131
1008 Zea mays DNA G636 131
1009 Zea mays DNA G636 131
1010 Zea mays DNA G636 131
1011 Pisum sativum PRT G636 131
1012 Glycine max DNA G638 133
1013 Glycine max DNA G638 133
1014 Glycine max DNA G638 133
1015 Glycine max DNA G638 133
1016 Medicago truncatula DNA G638 133
1017 Glycine max DNA G652 135
1018 Glycine max DNA G652 135
1019 Glycine max DNA G652 135
1020 Glycine max DNA G652 135
1021 Glycine max DNA G652 135
1022 Glycine max DNA G652 135
1023 Glycine max DNA G652 135
1024 Glycine max DNA G652 135
1025 Oryza sativa DNA G652 135
1026 Oryza sativa DNA G652 135
1027 Oryza sativa DNA G652 135
1028 Zea mays DNA G652 135
1029 Zea mays DNA G652 135
1030 Zea mays DNA G652 135
1031 Zea mays DNA G652 135
1032 Zea mays DNA G652 135
1033 Zea mays DNA G652 135
1034 Zea mays DNA G652 135
1035 Oryza sativa PRT G652 135
1036 Oryza sativa PRT G652 135
1037 Oryza sativa PRT G652 135
1038 Oryza sativa PRT G652 135
1039 Oryza sativa PRT G652 135
1040 Oryza sativa PRT G652 135
1041 Oryza sativa PRT G652 135
1042 Oryza sativa PRT G652 135
1043 Glycine max DNA G663 137
1044 Glycine max DNA G664 139
1045 Glycine max DNA G664 139
1046 Glycine max DNA G664 139
1047 Glycine max DNA G664 139
1048 Glycine max DNA G664 139
1049 Glycine max DNA G664 139
1050 Glycine max DNA G664 139
1051 Oryza sativa DNA G664 139
1052 Oryza sativa DNA G664 139
1053 Oryza sativa DNA G664 139
1054 Oryza sativa DNA G664 139
1055 Zea mays DNA G664 139
1056 Zea mays DNA G664 139
1057 Zea mays DNA G664 139
1058 Zea mays DNA G664 139
1059 Zea mays DNA G664 139
1060 Zea mays DNA G664 139
1061 Zea mays DNA G664 139
1062 Zea mays DNA G664 139
1063 G3509 Lycopersicon DNA G664 139
esculentum
1064 G3506 Oryza sativa PRT G664 139
1065 G3504 Oryza sativa PRT G664 139
1066 Oryza sativa PRT G664 139
1067 Oryza sativa PRT G664 139
1068 G3503 Oryza sativa indica PRT G664 139
1069 G3505 Oryza sativa japonica PRT G664 139
1070 G3507 Oryza sativa japonica PRT G664 139
1071 G3508 Oryza sativa japonica PRT G664 139
1072 G3509 Lycopersicon PRT G664 139
esculentum
1073 Hordeum vulgare PRT G664 139
subsp. vulgare
1074 Oryza sativa DNA G680 145
1075 Zea mays DNA G680 145
1076 Glycine max DNA G682 147
1077 Hordeum vulgare DNA G682 147
subsp. vulgare
1078 Populus tremula × DNA G682 147
Populus tremuloides
1079 Triticum aestivum DNA G682 147
1080 Gossypium arboreum DNA G682 147
1081 Oryza sativa PRT G682 147
1082 Oryza sativa PRT G682 147
1083 Glycine max PRT G682 147
1084 Glycine max PRT G682 147
1085 Glycine max PRT G682 147
1086 Glycine max PRT G682 147
1087 Glycine max PRT G682 147
1088 Glycine max PRT G682 147
1089 Zea mays PRT G682 147
1090 Zea mays PRT G682 147
1091 Glycine max DNA G715, G1646 149, 313
1092 Glycine max DNA G715, G1646 149, 313
1093 Glycine max DNA G715, G1646 149, 313
1094 Oryza sativa DNA G715, G1646 149, 313
1095 Oryza sativa DNA G715, G1646 149, 313
1096 Zea mays DNA G715, G1646 149, 313
1097 Zea mays DNA G715, G1646 149, 313
1098 Zea mays DNA G715, G1646 149, 313
1099 Zea mays DNA G715, G1646 149, 313
1100 Zea mays DNA G715, G1646 149, 313
1101 Zea mays DNA G715, G1646 149, 313
1102 Zea mays DNA G715, G1646 149, 313
1103 Zea mays DNA G715, G1646 149, 313
1104 Zea mays DNA G715, G1646 149, 313
1105 Oryza sativa PRT G715, G1646 149, 313
1106 Oryza sativa PRT G715, G1646 149, 313
1107 Oryza sativa PRT G715, G1646 149, 313
1108 Oryza sativa PRT G715, G1646 149, 313
1109 Oryza sativa PRT G715, G1646 149, 313
1110 Oryza sativa PRT G715, G1646 149, 313
1111 Glycine max DNA G720 151
1112 Glycine max DNA G720 151
1113 Glycine max DNA G720 151
1114 Glycine max DNA G720 151
1115 Medicago truncatula DNA G720 151
1116 Lycopersicon DNA G720 151
esculentum
1117 Lycopersicon DNA G720 151
esculentum
1118 Lycopersicon DNA G720 151
esculentum
1119 Solanum tuberosum DNA G720 151
1120 Glycine max DNA G736 153
1121 Glycine max DNA G736 153
1122 Oryza sativa PRT G736 153
1123 Glycine max DNA G748 155
1124 Glycine max DNA G748 155
1125 Glycine max DNA G748 155
1126 Oryza sativa DNA G748 155
1127 Oryza sativa DNA G748 155
1128 Zea mays DNA G748 155
1129 Oryza sativa PRT G748 155
1130 Oryza sativa PRT G748 155
1131 Oryza sativa PRT G748 155
1132 Oryza sativa PRT G748 155
1133 Cucurbita maxima PRT G748 155
1134 Glycine max DNA G789, G1494 159, 291
1135 Glycine max DNA G789, G1494 159, 291
1136 Oryza sativa DNA G789 159
1137 Oryza sativa DNA G789, G1494 159, 291
1138 Zea mays DNA G789, G1494 159, 291
1139 Oryza sativa PRT G789, G1494 159, 291
1140 Oryza sativa PRT G789, G1494 159, 291
1141 Oryza sativa PRT G789, G1494 159, 291
1142 Glycine max DNA G801 161
1143 Glycine max DNA G801 161
1144 Zea mays DNA G801 161
1145 Glycine max DNA G849 163
1146 Glycine max DNA G849 163
1147 Glycine max DNA G849 163
1148 Glycine max DNA G849 163
1149 Glycine max DNA G849 163
1150 Glycine max DNA G849 163
1151 Zea mays DNA G849 163
1152 Zea mays DNA G849 163
1153 Zea mays DNA G849 163
1154 Glycine max DNA G864 167
1155 Glycine max DNA G864 167
1156 Zea mays DNA G864 167
1157 Oryza sativa PRT G864 167
1158 Oryza sativa PRT G864 167
1159 Glycine max DNA G867, G1930 169, 369
1160 Glycine max DNA G867, G1930 169, 369
1161 Glycine max DNA G867, G1930 169, 369
1162 Glycine max DNA G867, G1930 169, 369
1163 Glycine max DNA G867, G1930 169, 369
1164 Glycine max DNA G867 169
1165 Oryza sativa DNA G867 169
1166 Oryza sativa DNA G867, G1930 169, 369
1167 Zea mays DNA G867, G1930 169, 369
1168 Zea mays DNA G867, G1930 169, 369
1169 Zea mays DNA G867, G1930 169, 369
1170 Zea mays DNA G867, G1930 169, 369
1171 Glycine max DNA G867, G1930 169, 369
1172 Mesembryanthemum DNA G867, G1930 169, 369
crystallinum
1173 Lycopersicon DNA G867, G1930 169, 369
esculentum
1174 Solanum tuberosum DNA G867, G1930 169, 369
1175 Hordeum vulgare DNA G867, G1930 169, 369
1176 Oryza sativa PRT G867, G1930 169, 369
1177 Oryza sativa PRT G867, G1930 169, 369
1178 Oryza sativa PRT G867, G1930 169, 369
1179 Oryza sativa PRT G867, G1930 169, 369
1180 Oryza sativa PRT G867, G1930 169, 369
1181 Oryza sativa PRT G867, G1930 169, 369
1182 Glycine max PRT G867, G1930 169, 369
1183 Glycine max PRT G867, G1930 169, 369
1184 Glycine max PRT G867, G1930 169, 369
1185 Zea mays PRT G867, G1930 169, 369
1186 Zea mays PRT G867, G1930 169, 369
1187 Glycine max DNA G869 171
1188 Glycine max DNA G869 171
1189 Oryza sativa DNA G869 171
1190 Zea mays DNA G869 171
1191 Oryza sativa PRT G869 171
1192 Oryza sativa DNA G877 173
1193 Glycine max DNA G881 175
1194 Oryza sativa DNA G881 175
1195 Oryza sativa DNA G881 175
1196 Zea mays DNA G881 175
1197 Zea mays DNA G881 175
1198 Zea mays DNA G881 175
1199 Zea mays DNA G881 175
1200 Oryza sativa PRT G881 175
1201 Oryza sativa PRT G892 177
1202 Mentha × piperita DNA G896 179
1203 Glycine max DNA G910 181
1204 Glycine max DNA G912 185
1205 Glycine max DNA G912 185
1206 Glycine max DNA G912 185
1207 Glycine max DNA G912 185
1208 Glycine max DNA G912 185
1209 Glycine max DNA G912 185
1210 Glycine max DNA G912 185
1211 Oryza sativa DNA G912 185
1212 Oryza sativa DNA G912, G913 185, 187
1213 Zea mays DNA G912 185
1214 Zea mays DNA G912 185
1215 Zea mays DNA G912, G913 185, 187
1216 Zea mays DNA G912 185
1217 Zea mays DNA G912 185
1218 Brassica napus DNA G912, G913 185, 187
1219 Solanum tuberosum DNA G912 185
1220 Descurainia sophia DNA G912 185
1221 Oryza sativa PRT G912 185
1222 Oryza sativa PRT G912, G913 185, 187
1223 Oryza sativa PRT G912, G913 185, 187
1224 Oryza sativa PRT G912 185
1225 Brassica napus PRT G912 185
1226 Nicotiana tabacum PRT G912 185
1227 Oryza sativa PRT G912 185
1228 Oryza sativa PRT G912 185
1229 Oryza sativa PRT G912 185
1230 Oryza sativa PRT G912 185
1231 Oryza sativa PRT G912 185
1232 Oryza sativa PRT G912 185
1233 Oryza sativa PRT G912 185
1234 Oryza sativa PRT G912 185
1235 Oryza sativa PRT G912 185
1236 Oryza sativa PRT G912 185
1237 Glycine max PRT G912 185
1238 Glycine max PRT G912 185
1239 Glycine max PRT G912 185
1240 Glycine max PRT G912 185
1241 Glycine max PRT G912 185
1242 Glycine max PRT G912 185
1243 Glycine max PRT G912 185
1244 Zea mays PRT G912 185
1245 Zea mays PRT G912 185
1246 Zea mays PRT G912 185
1247 Zea mays PRT G912 185
1248 Zea mays PRT G912 185
1249 Glycine max DNA G922 189
1250 Glycine max DNA G922 189
1251 Glycine max DNA G922 189
1252 Oryza sativa DNA G922 189
1253 Oryza sativa DNA G922 189
1254 Oryza sativa PRT G922 189
1255 Oryza sativa PRT G922 189
1256 Oryza sativa PRT G922 189
1257 Oryza sativa PRT G922 189
1258 Glycine max DNA G926 191
1259 Glycine max DNA G926 191
1260 Oryza sativa DNA G926 191
1261 Oryza sativa DNA G926 191
1262 Zea mays DNA G926 191
1263 Brassica napus PRT G926 191
1264 Glycine max DNA G961 193
1265 Glycine max DNA G961 193
1266 Oryza sativa DNA G961 193
1267 Zea mays DNA G961 193
1268 Zea mays DNA G961 193
1269 Zea mays DNA G961 193
1270 Oryza sativa PRT G961 193
1271 Glycine max DNA G974 197
1272 Glycine max DNA G974 197
1273 Glycine max DNA G974 197
1274 Glycine max DNA G974 197
1275 Glycine max DNA G974 197
1276 Glycine max DNA G974 197
1277 Oryza sativa DNA G974 197
1278 Zea mays DNA G974 197
1279 Zea mays DNA G974 197
1280 Zea mays DNA G974 197
1281 Zea mays DNA G974 197
1282 Lycopersicon DNA G974 197
esculentum
1283 Glycine max DNA G974 197
1284 Solanum tuberosum DNA G974 197
1285 Poplar xylem DNA G974 197
1286 Medicago truncatula DNA G974 197
1287 Sorghum bicolor DNA G974 197
1288 Oryza sativa PRT G974 197
1289 Oryza sativa PRT G974 197
1290 Oryza sativa PRT G974 197
1291 Atriplex hortensis PRT G974 197
1292 Glycine max DNA G975, G2583 199, 449
1293 Glycine max DNA G975, G2583 199, 449
1294 Glycine max DNA G975, G2583 199, 449
1295 Glycine max DNA G975, G2583 199, 449
1296 Glycine max DNA G975, G2583 199, 449
1297 Oryza sativa DNA G975 199
1298 Oryza sativa DNA G975, G2583 199, 449
1299 Zea mays DNA G975, G2583 199, 449
1300 Zea mays DNA G975, G2583 199, 449
1301 Brassica rapa DNA G975, G2583 199, 449
1302 Oryza sativa PRT G975, G2583 199, 449
1303 Glycine max DNA G979 201
1304 Glycine max DNA G979 201
1305 Glycine max DNA G979 201
1306 Oryza sativa DNA G979 201
1307 Zea mays DNA G979 201
1308 Zea mays DNA G979 201
1309 Zea mays DNA G979 201
1310 Oryza sativa PRT G979 201
1311 Oryza sativa PRT G979 201
1312 Oryza sativa PRT G979 201
1313 Oryza sativa PRT G979 201
1314 Oryza sativa PRT G979 201
1315 Glycine max DNA G987 203
1316 Glycine max DNA G987 203
1317 Glycine max DNA G987 203
1318 Glycine max DNA G987 203
1319 Glycine max DNA G987 203
1320 Glycine max DNA G987 203
1321 Oryza sativa DNA G987 203
1322 Oryza sativa DNA G987 203
1323 Zea mays DNA G987 203
1324 Oryza sativa PRT G987 203
1325 Oryza sativa PRT G988 205
1326 Oryza sativa PRT G988 205
1327 Capsella rubella PRT G988 205
1328 Glycine max DNA G1040 207
1329 Glycine max DNA G1040 207
1330 Glycine max DNA G1040 207
1331 Glycine max DNA G1040 207
1332 Glycine max DNA G1040 207
1333 Zea mays DNA G1040 207
1334 Zea mays DNA G1040 207
1335 Zea mays DNA G1040 207
1336 Zea mays DNA G1040 207
1337 Zea mays DNA G1040 207
1338 Oryza sativa PRT G1040 207
1339 Oryza sativa PRT G1040 207
1340 Glycine max DNA G1047 209
1341 Zea mays DNA G1047 209
1342 Oryza sativa PRT G1047 209
1343 Oryza sativa PRT G1047 209
1344 Glycine max DNA G1051, G1052 211, 213
1345 Glycine max DNA G1051, G1052 211, 213
1346 Glycine max DNA G1051, G1052 211, 213
1347 Glycine max DNA G1051, G1052 211, 213
1348 Glycine max DNA G1051, G1052 211, 213
1349 Glycine max DNA G1051, G1052 211, 213
1350 Glycine max DNA G1051, G1052 211, 213
1351 Oryza sativa DNA G1051, G1052 211, 213
1352 Zea mays DNA G1051, G1052 211, 213
1353 Zea mays DNA G1051, G1052 211, 213
1354 Zea mays DNA G1051, G1052 211, 213
1355 Zea mays DNA G1051, G1052 211, 213
1356 Zea mays DNA G1051, G1052 211, 213
1357 Zea mays DNA G1051, G1052 211, 213
1358 Zea mays DNA G1051, G1052 211, 213
1359 Oryza sativa DNA G1052 213
1360 Zea mays DNA G1052 213
1361 Zea mays DNA G1052 213
1362 Oryza sativa PRT G1051, G1052 211, 213
1363 Oryza sativa PRT G1051, G1052 211, 213
1364 Oryza sativa PRT G1051, G1052 211, 213
1365 Glycine max DNA G1062 215
1366 Glycine max DNA G1062 215
1367 Glycine max DNA G1062 215
1368 Glycine max DNA G1062 215
1369 Oryza sativa DNA G1062 215
1370 Oryza sativa DNA G1062 215
1371 Zea mays DNA G1062 215
1372 Zea mays DNA G1062 215
1373 Zea mays DNA G1062 215
1374 Zea mays DNA G1062 215
1375 Zea mays DNA G1062 215
1376 Medicago truncatula DNA G1062 215
1377 Lycopersicon DNA G1062 215
esculentum
1378 Oryza sativa PRT G1062 215
1379 Glycine max DNA G1063, G2143 217, 413
1380 Glycine max DNA G1063, G2143 217, 413
1381 Glycine max DNA G1063, G2143 217, 413
1382 Glycine max DNA G1063, G2143 217, 413
1383 Glycine max DNA G1063, G2143 217, 413
1384 Lycopersicon DNA G1063, G2143 217, 413
esculentum
1385 Glycine max DNA G1064 219
1386 Glycine max DNA G1064 219
1387 Glycine max DNA G1064 219
1388 Zea mays DNA G1064 219
1389 Zea mays DNA G1064 219
1390 Lycopersicon DNA G1064 219
esculentum
1391 Oryza sativa PRT G1064 219
1392 Gossypium hirsutum PRT G1064 219
1393 Glycine max DNA G1069 221
1394 Glycine max DNA G1069 221
1395 Oryza sativa PRT G1069, G1073 221, 223
1396 Zea mays DNA G1069 221
1397 Lotus japonicus DNA G1069 221
1398 Lycopersicon DNA G1073 223
esculentum
1399 Oryza sativa PRT G1073 223
1400 Oryza sativa PRT G1073 223
1401 Oryza sativa PRT G1073 223
1402 Oryza sativa PRT G1073 223
1403 Oryza sativa PRT G1073 223
1404 Oryza sativa PRT G1073 223
1405 Oryza sativa PRT G1073 223
1406 Oryza sativa PRT G1073 223
1407 Oryza sativa PRT G1073 223
1408 Oryza sativa PRT G1073 223
1409 Oryza sativa PRT G1073 223
1410 Oryza sativa PRT G1073 223
1411 Glycine max PRT G1073 223
1412 Glycine max PRT G1073 223
1413 Glycine max PRT G1073 223
1414 Glycine max PRT G1073 223
1415 Glycine max PRT G1073 223
1416 Glycine max PRT G1073 223
1417 Glycine max PRT G1073 223
1418 Zea mays PRT G1073 223
1419 Glycine max DNA G1075 225
1420 Glycine max DNA G1075 225
1421 Glycine max DNA G1075 225
1422 Glycine max DNA G1075 225
1423 Glycine max DNA G1075 225
1424 Oryza sativa DNA G1075 225
1425 Oryza sativa DNA G1075 225
1426 Oryza sativa DNA G1075 225
1427 Oryza sativa DNA G1089 229
1428 Zea mays DNA G1089 229
1429 Zea mays DNA G1089 229
1430 Zea mays DNA G1089 229
1431 Zea mays DNA G1089 229
1432 Zea mays DNA G1089 229
1433 Oryza sativa PRT G1089 229
1434 Glycine max DNA G1134, G2555 231, 445
1435 Glycine max DNA G1134, G2555 231, 445
1436 Oryza sativa DNA G1134, G2555 231, 445
1437 Glycine max DNA G1140 233
1438 Glycine max DNA G1140 233
1439 Glycine max DNA G1140 233
1440 Glycine max DNA G1140 233
1441 Glycine max DNA G1140 233
1442 Glycine max DNA G1140 233
1443 Oryza sativa DNA G1140 233
1444 Zea mays DNA G1140 233
1445 Zea mays DNA G1140 233
1446 Zea mays DNA G1140 233
1447 Zea mays DNA G1140 233
1448 Zea mays DNA G1140 233
1449 Zea mays DNA G1140 233
1450 Zea mays DNA G1140 233
1451 Zea mays DNA G1140 233
1452 Zea mays DNA G1140 233
1453 Oryza sativa PRT G1140 233
1454 Ipomoea batatas PRT G1140 233
1455 Zea mays DNA G1146 237
1456 Zea mays DNA G1146 237
1457 Oryza sativa PRT G1146 237
1458 Oryza sativa PRT G1146 237
1459 Oryza sativa PRT G1146 237
1460 Glycine m